Anti-OX40 antibodies and methods of using the same

ABSTRACT

Human antibodies, preferably recombinant human antibodies, both humanized and chimeric, which specifically bind to human OX40 are disclosed. Preferred antibodies have high affinity for OX40 receptor and activate the receptor in vitro and in vivo. The antibody can be a full-length antibody or an antigen-binding portion thereof. The antibodies, or antibody portions, are useful for modulating receptor activity, e.g., in a human subject suffering from a disorder in which OX40 activity is detrimental. Nucleic acids, vectors and host cells for expressing the recombinant human antibodies are provided, and methods of synthesizing the recombinant human antibodies, are also provided.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R01AI061645-01,R01 AI062888-01, and U19 AI071130-01 awarded by the National Institutesof Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national phase application under 35 U.S.C. 371 ofInternational Application No. PCT/US2011/048752, filed Aug. 23, 2011,which claims the benefit of and priority in U.S. Patent ProvisionalApplication Ser. No. 61/375,999, filed on Aug. 23, 2010, and U.S. PatentProvisional Application Ser. No. 61/380,827, filed on Sep. 8, 2010. Theentire text of each of the above-referenced disclosures is specificallyherein incorporated by reference.

FIELD OF INVENTION

This invention relates generally to modulation of the OX40-receptoractivation, and more particularly, to modulating the OX40-receptor toinhibit the immunosuppressive function of Interleukin 10 (IL-10)producing CD4⁺ type 1 regulatory T cells (“Tr1 cells”) andFoxp3⁺-expressing regulatory T cells (also sometimes referred to hereinas “Foxp3⁺ T-reg” cells), and the generation of Tr1 cells from CD4⁺cells or naïve cells and IL-10 production.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

None.

REFERENCE TO SEQUENCE LISTING

This disclosure includes a sequence listing submitted as a text filepursuant to 37 C.F.R. §1.52(e)(v) named sequence listing.txt, created onAug. 23, 2011, with a size of 13,836 bytes, which is incorporated hereinby reference. The attached sequence descriptions and Sequence Listingcomply with the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984). The symbols and format used for nucleotide andamino acid sequence data comply with the rules set forth in 37 C.F.R.§1.822.

BACKGROUND OF THE INVENTION

Tr1 cells have a critical role in peripheral tolerance. Tr1 cells areparticularly important in limiting tissue damage to the host duringinflammatory immune responses. The generation of Tr1 cells accompaniesboth TH1 and TH2 immune responses in vivo and in vitro.

Tr1 cells are generated from naïve CD4⁺ T cells during an antigen-drivenT cell immune response. Tr1 cells are anergic in response to signalingthrough TCR, CD28 and IL-2 receptors and have the ability to suppressantigen-driven proliferation of naïve CD4⁺ T cells in vivo and in vitro.Tr1 cells have the ability to inhibit the development of autoimmunediseases and limit the magnitude of immune responses to microbialpathogens.

While the molecular signals that lead to the Tr1 cells have beenstudied, little is known about the molecular signals that negativelyregulate the generation of these cells. Although immunosuppressivedrugs, cytokines, co-stimulatory molecules, and DCs have been implicatedin the induction of Tr1 cells, signals that negatively regulate thegeneration of Tr1 cells remain elusive.

BRIEF SUMMARY OF THE INVENTION

Activation of the OX40 receptor blocks Tr1 generation from naïve ormemory CD4⁺ T cells as well as IL-10 production from Tr1 cells and theimmunosuppressive function of the Tr1 cells. Activation of the OX40receptor also blocks IL-10 production by Foxp3⁺ T-reg cells andimmunosuppressive function. As such, presented herein are agonistantibodies that bind to the OX40 receptor, whereby the agonist modulatesthe activation of the OX40 receptor to block IL-10 cytokine secretionand/or the Tr1 and Foxp3⁺ T-reg cells overall immunosuppressivefunction. Essentially, the antibodies can mimic the OX40 ligand andtrigger the OX40 receptor on Tr1 and/or on natural T regulatory cells(“nTregs”), also referred to as “Foxp3⁺ T-regs.”

As we shown in co-pending patent application U.S. Ser. Nos. 11/659,266and 12/861,135, OX40L inhibits the generation and function ofIL-10-producing Tr1 cells from naïve and memory CD4+ T cells that wereinduced by the immunosuppressive drugs dexamethasone and vitamin D3. Wediscovered that OX40L inhibits the generation and function of IL-10producing regulatory T cells. These discoveries demonstrate thatsignaling OX40 by OX40L suppresses the generation of human IL-10producing immunosuppressive T cells in culture. This unique function ofOX40L is not shared by two other co-stimulatory TNF-family members,GITR-ligand and 4-1BB-ligand. OX40L also strongly inhibits thegeneration and function of IL-10-producing Tr1 cells induced by twophysiological stimuli provided by inducible co-stimulator ligand andimmature DCs. Signaling the OX40 receptor on human T cells by monoclonalantibodies, small molecules, or by the OX40L, or protein having at least90 percent homology thereto, modulates and regulates the generation andfunction of IL-10 producing immunosuppressive T cells.

The discovery lends to numerous applications of treatment. For example,agonistic antibodies, small molecules, or OX40L could be used tosuppress the generation and the function of IL-10 producingimmunosuppressive T cells and therefore could be used to enhance immuneresponses to treat cancer and infectious diseases, or as an adjuvant forcancer vaccines. Antagonistic antibodies to OX40 or to OX40L, orantagonistic small molecules, could be used to enhance the generationand the function of IL-10-producing immunosuppressive T cells andtherefore could be used for the development of therapies for autoimmunediseases and graft versus host diseases. Our discovery also provides forhigh throughput methods for screening antibodies or small moleculeseither activating the OX40 receptor (or conversely blocking OX40signaling) on T cells for the development of therapeutics for cancer, oralternatively, autoimmune diseases, and graft versus host diseases.

Monoclonal and human antibodies (sometimes referred to herein as an“anti-OX40 antibody” and/or other variations of the same) that bindhuman OX40 receptor are provided herein. These antibodies are useful inthe treatment or prevention of acute or chronic diseases or conditionswhose pathology involves OX40. In one aspect, an isolated humanantibody, or an antigen-binding portion thereof, that binds to humanOX40 and is effective as a cancer treatment or treatment against anautoimmune disease is described. Any of the anti-OX40 antibodiesdisclosed herein may be used as a medicament. Any one or more of theanti-OX40 antibodies may be used to treat one or more a variety ofcancers or autoimmune disease described herein.

Isolated humanized antibodies that bind to OX40 are provided herein. Theisolated antibodies as described herein bind to OX40, and may bind toOX40 encoded from the following genes: NCBI Accession NumberNP_(—)003317, Genpept Accession Number P23510, or genes having 90percent homology thereto. The isolated antibody provided herein mayfurther bind to the OX40 receptor having one of the following GenBankAccession Numbers: AAB39944, CAE11757, or AAI05071.

As taught herein, exemplary is an isolated antibody which binds to OX40comprising: (a) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (b) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (c) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO. 3;(d) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO. 7; (e) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO. 8; and (f) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO. 9.

Furthermore, another example is an isolated antibody which binds to OX40comprising: (a) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 13; (b) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 14; (c) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO.15; (d) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO. 19; (e) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO, 20; and (f) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO. 21.

Alternatively, an isolated antibody may have a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 13; aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 2 or 14; and/or a heavy chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 3 or 15, or a heavy chain variableregion CDR having 90 percent homology thereto.

Further, an isolated antibody may have a light chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 7 or 19; a lightchain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 8 or 20 and/or a light chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 9 or 21, or a heavy chain variableregion having 90 percent homology thereto.

The isolated antibody may have a light chain variable region (“VL”)comprising the amino acid sequence of SEQ ID NO: 10, 11, 22 or 23, or anamino acid sequence with at least 90 percent identity to the amino acidsequences of SEQ ID NO: 10, 11, 22 or 23. The isolated antibody may havea heavy chain variable region (“VH”) comprising the amino acid sequenceof SEQ ID NO: 4, 5, 16 and 17, or an amino acid sequence with at least90 percent identity to the amino acid sequences of SEQ ID NO: 4, 5, 16and 17. As such, as an example, the isolated antibody may comprise avariable heavy sequence of SEQ ID NO:5 and a variable light sequence ofSEQ ID NO: 11, or a sequence having 90 percent homology thereto.Similarly, the isolated antibody can have a variable heavy sequence ofSEQ ID NO:17 and a variable light sequence of SEQ ID NO: 23 or asequence having 90 percent homology thereto.

The isolated antibody may have variable light chain encoded by thenucleic acid sequence of SEQ ID NO: 12, or 24, or a nucleic acidsequence with at least 90 percent identity to the nucleotide sequencesof SEQ ID NO: 12 or 24. The isolated antibody may have variable heavychain encoded by a nucleic acid sequence of SEQ ID NO: 6 or 18, or anucleic acid sequence with at least 90 percent identity to nucleotidesequences of SEQ ID NO: 6 or 18.

Also provided herein are monoclonal antibodies. The monoclonalantibodies may have a variable light chain comprising the amino acidsequence of SEQ ID NO: 10 or 22, or an amino acid sequence with at least90 percent identity to the amino acid sequences of SEQ ID NO: 10 or 22.Further provided are monoclonal antibodies having a variable heavy chaincomprising the amino acid sequence of SEQ ID NO: 4 or 16, or an aminoacid sequence with at least 90 percent identity to the amino acidsequences of SEQ ID NO: 4 or 16.

Also provided herein is isolated nucleic acid encoding any of theanti-OX40 antibodies taught herein. Further provided herein are hostcells, each comprising nucleic acid encoding any of the anti-OX40antibodies described herein. Methods of producing an antibody (such asthe host cell comprising nucleic acid encoding any of the anti-OX40antibodies described herein) comprising culturing the host cell so thatthe antibody is produced, and/or recovering the antibody from the hostcell, are further provided.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above-recited features, aspects andadvantages of the invention, as well as others that will becomeapparent, are attained and can be understood in detail, more particulardescription of the invention briefly summarized above can be had byreference to the embodiments thereof that are illustrated in thedrawings that form a part of this specification. It is to be noted,however, that the appended drawings illustrate some embodiments of theinvention and are, therefore, not to be considered limiting of theinvention's scope, for the invention can admit to other equallyeffective embodiments.

FIG. 1 shows that FOXP3⁺ Tregs infiltrated human follicular lymphoma(FL) tissues and co-localized with tumor B cells and monocytes. Left:Double immunostaining of FOXP3⁺ Tregs (red) and CD20⁺ B lymphoma cells(green); Right: FOXP3⁺ Tregs (red) and CD11c⁺ monocytes/macrophage/DC(green).

FIGS. 2A and 2B show increased numbers of CD4+FOXP3⁺ Tregs in patientswith FL. Tumor cells and PBMCs were obtained from six patients with FLat initial diagnosis before therapy. PBMCs were also obtained from sixnormal donors for comparison. The percentages of regulatory T cells overtotal CD4⁺ T cells were determined by flow cytometric analysis ofCD4⁺CD25⁺CD127^(low)FOXP3⁺ Tregs. FIG. 2A shows representative FACSanalysis of Tregs. FL PBMC and FL tumor cells were divided from the samepatient. FIG. 2B shows the percentage of Tregs of all donors. Horizontalbar indicate means.

FIG. 3 shows the isolation of ICOS⁺FOXP3⁺ or ICOS⁻FOXP3⁺ Tregs from FL.Single cell suspension was obtained from a spleen specimen before anytreatment. Cells were thawed on the day of assay. EnrichedCD4⁺CD8⁻CD14⁻CD16⁻CD56⁻CD11c⁻TCRγδ⁻ T cells were divided into CD25^(low)and CD25^(high) subsets. CD4⁺CD25^(high)FOXP3⁺ Tregs were further sortedinto ICOS^(high) and ICOS^(low) subsets based on surface expression ofICOS. Intracellular expression of FOXP3 was determined in all subsets.

FIG. 4 shows intratumoral Tregs inhibit proliferation of infiltratingCD4⁺CD25⁻ T cells in FL, and the inhibition could be partially blockedby anti-IL-10 neutralization antibodies. CFSE-labeled CD4⁺CD25⁻tumor-infiltrating T cells were cultured with autologous tumor cellspreactivated by recombinant CD40L in the presence or absence ofautologous ICOS⁺FOXP3⁺ Tregs or ICOS⁻FOXP3⁺ Tregs, or anti-IL-10 (10μg/ml). After 72 hours of culture, proliferation of CD4⁺CD25⁻ cells wasdetermined by flow cytometric analysis of CFSE dilution.

FIG. 5A shows the intracellular analysis of cytokine production by naïveCD4⁺ T cells as determined by flow cytometry according to an embodimentof a method of the present invention.

FIG. 5B shows cytokine production by naïve CD4⁺ T cells as determined byELISA according to an embodiment of a method of the present invention.

FIG. 5C shows suppressive function by IL-10-producing Tr1 cells asdetermined by [³H]thymidine incorporation according to an embodiment ofa method of the present invention.

FIG. 6A shows the intracellular analysis of cytokine production bymemory CD4⁺ T cells as determined by flow cytometry according to anembodiment of a method of the present invention.

FIG. 6B shows IL-10 production by memory CD4⁺ T cells as determined byELISA according to an embodiment of a method of the present invention.

FIG. 7A shows the intracellular analysis of cytokine production by naïveCD4⁺ T cells as determined by flow cytometry according to an embodimentof a method of the present invention.

FIG. 7B shows IL-10 production by naïve CD4⁺ T cells as determined byELISA according to an embodiment of a method of the present invention.

FIG. 7C shows the number of viable T cells counted according to anembodiment of a method of the present invention.

FIG. 8A shows the intracellular analysis of cytokine production by naïveCD4⁺ T cells as determined by flow cytometry according to an embodimentof a method of the present invention.

FIG. 8B shows IL-10 production by naïve CD4⁺ T cells as determined byELISA according to an embodiment of a method of the present invention.

FIG. 8C shows the intracellular analysis of cytokine production bymemory CD4⁺ T cells as determined by flow cytometry according to anembodiment of a method of the present invention.

FIG. 8D shows IL-10 production by memory CD4⁺ T cells as determined byELISA according to an embodiment of a method of the present invention.

FIG. 8E shows the intracellular analysis of cytokine production by naïveCD4⁺ T cells as determined by flow cytometry according to an embodimentof a method of the present invention.

FIG. 8F shows IL-10 production by naïve CD4⁺ T cells as determined byELISA according to an embodiment of a method of the present invention.

FIG. 9 shows IL-10 production by regulatory T cells as determined byELISA according to an embodiment of a method of the present invention.

FIG. 10 shows the results of screening of anti-human OX40 hybridomasupernatants against L-OX40 versus L parental cells as determined byELISA.

FIG. 11 shows the screening of human OX40-specific monoclonal antibodiesas determined by flow cytometry analysis according to an embodiment of amethod of the present invention.

FIG. 12 shows the confirmation of anti-hOX40 monoclonal antibodiesspecificity by using SUPM2 cells expressing OX40 (SUPM2-OX40) accordingto an embodiment of a method of the present invention.

FIG. 13 shows OX40-specific monoclonal antibodies that can inhibit thegeneration of IL-10 producing cells (Tr1) from CD4⁺ T cells stimulatedby vit D₃ (0.1 μM)/Dex (50 nm), CD32L/ICOSL and anti-CD3/CD28 (0.2μg/ml) according to an embodiment of a method of the present invention.Representative Fluorescence Activated Cell Sorting (FACS) data are shownin A and the percentages of IL-10 producing cells for all OX40monoclonal antibodies treatments are shown in B.

FIG. 14 shows the results of hOX40-specific monoclonal antibodies thatinhibit Tr1 cell generation also stimulate CD4⁺ T cell proliferationaccording to an embodiment of a method of the present invention.

FIGS. 15A and 15B details the titration of OX40 monoclonal antibodiesfor their ability to inhibit the generation of Tr1 cells from CD4⁺ Tcells according to an embodiment of a method of the present invention.Representative FACS data are shown in FIG. 15A and percentage of Tr1cells after treatment with nine OX40 monoclonal antibodies are shown inFIG. 15B.

FIGS. 16A, 16B, and 16C shows OX40-specific monoclonal antibodies thatinhibit IL-10 producing Tr1 cell generation from CD4⁺ T cells alsoinhibit ICOS⁺CD4⁺CD25^(high)CD127⁻ Treg IL-10 production andimmunosuppressive function. Freshly sorted ICOS⁺CD4⁺CD25^(high)CD127⁻Tregs (ICOS⁺Tregs) were stimulated with anti-CD3 (0.2 μg/ml) in thepresence of CD32L/ICOSL cells and CD32L/OX40L cells (FIG. 16A) or OX40monoclonal antibodies or control antibody (FIG. 16B) for five days.Cells were then restimulated with anti-CD3/CD28 for 24 hours and thesupernatants were assayed for IL-10 by enzyme-linked immunosorbent assay(ELISA). FIG. 16C is a monocyte-based proliferation assay showing thattwo of the antibodies blocked ICOS⁺Treg function.

FIGS. 17A and 17B shows the identification of anti-hOX40 monoclonalantibodies that inhibit the generation of Tr1 cells and blockFOXP3⁺CD4⁺CD25^(high) Treg function according to an embodiment of amethod of the present invention. Representative flow cytometry analysesare shown in FIG. 17A. Data for six monoclonal antibodies are shown inFIG. 17B.

FIG. 18 demonstrates the identification of anti-hOX40 monoclonalantibodies that do not inhibit Tr1 cell generation but blockFOXP3⁺CD4⁺CD25^(high) Treg function according to an embodiment of amethod of the present invention.

FIGS. 19A and 19B shows anti-hOX40 agonist antibodies blockinglymphoma-derived CD4⁺CD25^(high) Treg function according to anembodiment of a method of the present invention. Representative FACSanalyses are shown in FIG. 19A and data for all experiments are shown inFIG. 19B.

FIG. 20 shows that anti-hOX40 monoclonal antibodies can bind to rhesusCD4⁺ T cells. As shown, six of the anti-hOX40 mAbs can bind to rhesusactivated CD4⁺ T cells and will bind to rhesus OX40 and activate OX40signaling.

FIG. 21 shows that each of Hu106-222 Lot I and II antibodies of ExampleI is comprised of a heavy chain with a molecular weight of about 50 kDand a light chain with a molecular weight of about 25 kD. The purity ofHu106-222 Lot I and II antibodies appeared to be more than 95%.

FIG. 22 shows the analysis of mouse 106-122, Ch106 and Hu106-222 (LotII) antibodies for binding to L/OX40 cells (Example I).

FIG. 23 depicts the schematic structure of the expression vector forHu106 IgG1/kappa antibody (Expression Vector). Proceeding clockwise fromthe SalI site at the top, the plasmid contains the heavy chaintranscription unit starting with the human cytomegalovirus (CMV) majorimmediate early promoter and enhancer (CMV promoter) to initiatetranscription of the antibody heavy chain gene. The CMV promoter isfollowed by the VH exon, a genomic sequence containing the human gamma-1heavy chain constant region including the CH1, hinge, CH2 and CH3 exonswith the intervening introns, and the polyadenylation site following theCH3 exon. After the heavy chain gene sequence, the light chaintranscription unit begins with the CMV promoter, followed by the VL exonand a genomic sequence containing the human kappa chain constant regionexon (CL) with pa-t of the intron preceding it, and the polyadenylationsite following the CL exon. The light chain gene is then followed by theSV40 early promoter (SV40 promoter), the E. coli xanthine guaninephosphoribosyl transferase gene (gpt), and a segment containing the SV40polyadenylation site (SV40 poly(A) site). Finally, the plasmid containsa part of the plasmid pUC19, comprising the bacterial origin ofreplication (pUC ori) and beta-lactamase gene (beta lactamase).Locations of relevant restriction enzyme sites are shown in the figure.

FIG. 24 shows the comparison between Hu 106-222 Lot I and II antibodiesfor binding to L/OX40 cells (Example I below).

FIG. 25 shows Hu119-122 is comprised of a heavy chain with a molecularweight of about 50 kD and a light chain with a molecular weight of about25 kD. The purity of Hu119 appeared to be more than 95% (Example IIbelow).

FIG. 26 shows the result of the FACS analysis of Ch119-122 andHul119-122 antibodies described herein (Example II below).

FIG. 27 shows that humanized anti-human OX40 mAb clone 119-122 (Hu119),and its FcR binding mutated antibody (Hu119-AA) enhanced naïve CD4⁺ Tcell proliferation. Hu119-122 yielded better T cell stimulatory activitycompared to parental mouse anti-human OX40 mAb (Mouse119-122). However,chimeric anti-human OX40 mAb (Ch119, mouse VH and VL but human gamma-1and kappa constant regions) failed to enhance T cell proliferation.

FIG. 28 shows FcR binding mutated humanized anti-human OX40 mAb clone106-222 (Hu222AA) and chimeric anti-human OX40 mAb clone 106-222 (Ch222)enhanced anti-CD3 stimulated naïve CD4⁺ T cell proliferation. Theseantibodies have similar stimulatory activity compared to parental mouseanti-human OX40 mAb (Mouse222). However, the fully humanized anti-humanOX40 Ab, Hu222, did not enhance T cell proliferation compared to humanIgG1.

FIGS. 29A and B shows that the humanized and mouse anti-human OX40 mAbclone 119-122 blocks CD4⁺ Treg suppressive function.

FIG. 30 provides data showing anti-human OX40 antibodies enhance CD4⁺and CD8⁺ T cell proliferation using plate-bound antibodies.

FIG. 31 shows humanized and mouse anti-human OX40 antibodies requirecross-linking in order to enhance T cell proliferation.

FIG. 32 shows anti-human OX40 antibodies block the activity ofCD4⁺FOXP3⁺nTregs using plate-bound antibodies.

FIG. 33 shows that a high concentration of mouse anti-human OX40antibodies preferentially kills FOXP3⁺ Tregs.

FIG. 34 shows mouse anti-human OX40 mAbs act directly on either effectorT cells or nTregs to block the suppressive function of Tregs.

FIGS. 35A, 35B, and 35C show the results of anti-hOX40 mAb tumortreatment in mice adaptively transferred with hOX40⁺CD8⁺ T cells. Theanti-human OX40 mAb promotes T cell expansion and survival in vivo. Thetherapeutic vaccination regimen is shown in FIG. 35A. Representative invivo bioluminescence images are shown in FIG. 35B. Results of theantibody tumor treatment are shown in FIG. 35C.

FIG. 36 shows the alignment of the amino acid sequences of 106-222,humanized 106-222 (Hu106), and human acceptor X61012 (GenBank accessionnumber) VH sequences are shown. Amino acid residues are shown in singleletter code. Numbers above the sequences indicate the locationsaccording to Kabat et al. (Sequences of Proteins of ImmunologicalInterests, Fifth edition, NIH Publication No. 91-3242, U.S. Departmentof Health and Human Services, 1991). The same sequences as claimedherein are also provided in the Sequence Listing and the positionnumbers may be different. In FIG. 36, CDR sequences defined by Kabat etal. (1991) are underlined in 106-222 VH. CDR residues in X61012 VH areomitted in the figure. Human VH sequences homologous to the 106-222 VHframeworks were searched for within the GenBank database, and the VHsequence encoded by the human X61012 cDNA (X61012 VH) was chosen as anacceptor for humanization. The CDR sequences of 106-222 VH were firsttransferred to the corresponding positions of X61012 VH. Next, atframework positions where the three-dimensional model of the 106-222variable regions suggested significant contact with the CDRs, amino acidresidues of mouse 106-222 VH were substituted for the correspondinghuman residues. These substitutions were performed at positions 46 and94 (underlined in Hu106 VH). In addition, a human framework residue thatwas found to be atypical in the corresponding V region subgroup wassubstituted with the most typical residue to reduce potentialimmunogenicity. This substitution was performed at position 105(double-underlined in Hu106 VH).

FIG. 37 shows alignment of the amino acid sequences of 106-222,humanized 106-222 (Hu106), and human acceptor AJ388641 (GenBankaccession number) VL sequences is shown. Amino acid residues are shownin single letter code. Numbers above the sequences indicate thelocations according to Kabat et al. (1991). The same sequences asclaimed herein are also provided in the Sequence Listing although theposition numbers may be different, CDR sequences defined by Kabat et al.(1) are underlined in 106-222 VH. CDR residues in AJ388641 VL areomitted in the figure. Human VL sequences homologous to the 106-222 VLframeworks were searched for within the GenBank database, and the VLsequence encoded by the human AJ388641 cDNA (AJ388641 VL) was chosen asan acceptor for humanization. The CDR sequences of 106-222 VL weretransferred to the corresponding positions of AJ388641 VL. No frameworksubstitutions were performed in the humanized form.

FIG. 38 shows the nucleotide sequence of the Hu106 VH gene flanked bySpeI and HindIII sites (underlined) is shown along with the deducedamino acid sequence. Amino acid residues are shown in single lettercode. The signal peptide sequence is in italic. The N-terminal aminoacid residue (Q) of the mature VH is double-underlined. CDR sequencesaccording to the definition of Kabat et al. (1991) are underlined. Thesame sequences as claimed herein are also provided in the SequenceListing and the position numbers may be different in the SequenceListing. The intron sequence is in italic. Hu106 VH gene fragmentdigested with SpeI and HindIII was cloned between the correspondingsites in the Expression Vector shown in FIG. 23.

FIG. 39 shows the nucleotide sequence of the Hu106-222 VL gene flankedby NheI and EcoRI sites (underlined) is shown along with the deducedamino acid sequence. Amino acid residues are shown in single lettercode. The signal peptide sequence is in italic. The N-terminal aminoacid residue (D) of the mature VL is double-underlined. CDR sequencesaccording to the definition of Kabat et al. (1991) are underlined. Theintron sequence is in italic. Hu106 VL gene fragment digested with NheIand EcoRI was cloned between the corresponding sites in the ExpressionVector shown in FIG. 23. The same sequences as claimed herein are alsoprovided in the Sequence Listing although the position numbers may bedifferent in the Sequence Listing.

FIG. 40 shows the alignment of the amino acid sequences of 119-122,humanized 119-122 (Hu119), and human acceptor Z14189 (GenBank accessionnumber) VH sequences are shown. Amino acid residues are shown in singleletter code. Numbers above the sequences indicate the locationsaccording to Kabat et al. (Sequences of Proteins of ImmunologicalInterests, Fifth edition, NIH Publication No. 91-3242, U.S. Departmentof Health and Human Services, 1991). CDR sequences defined by Kabat etal. (1991) are underlined in 119-122 VH. CDR residues in Z14189 VH areomitted in the figure. Human VH sequences homologous to the 119-122 VHframeworks were searched for within the GenBank database, and the VHsequence encoded by the human Z14189 cDNA (Z14189 VH) was chosen as anacceptor for humanization. The CDR sequences of 119-122 VH were firsttransferred to the corresponding positions of Z14189 VH. Next, atframework positions where the three-dimensional model of the 119-122variable regions suggested significant contact with the CDRs, amino acidresidues of mouse 119-122 VH were substituted for the correspondinghuman residues. These substitutions were performed at positions 26, 27,28, 30 and 47 (underlined in the Hul1119 VH sequence) as shown on thefigure. The same sequences as claimed herein are also provided in theSequence Listing although the position numbers may be different in theSequence Listing.

FIG. 41 shows the alignment of the amino acid sequences of 119-122,humanized 119-122 (Hu119), and human acceptor M29469 (GenBank accessionnumber) VL sequences are shown. Amino acid residues are shown in singleletter code. Numbers above the sequences indicate the locationsaccording to Kabat et al. (1991). CDR sequences defined by Kabat et al.(1) are underlined in 119-122 VL. CDR residues in M29469 VL are omittedin the sequence. Human VL sequences homologous to the 119-122 VLframeworks were searched for within the GenBank database, and the VLsequence encoded by the human M29469 cDNA (M29469 VL) was chosen as anacceptor for humanization. The CDR sequences of 119-122 VL weretransferred to the corresponding positions of M29469 VL. No frameworksubstitutions were needed in the humanized form. The same sequences asclaimed herein are also provided in the Sequence Listing although theposition numbers may be different in the Sequence Listing.

FIG. 42 shows the nucleotide sequence of the Hun 19 VH gene flanked bySpeI and HindIII sites (underlined) is shown along with the deducedamino acid sequence. Amino acid residues are shown in single lettercode. The signal peptide sequence is in italic. The N-terminal aminoacid residue (E) of the mature VH is double-underlined. CDR sequencesaccording to the definition of Kabat et al. (1991) are underlined. Theintron sequence is in italic. Hul19 VH gene fragment digested with SpeIand HindIII was cloned between the corresponding sites in the ExpressionVector shown in FIG. 23. The same sequences as claimed herein are alsoprovided in the Sequence Listing although the position numbers may bedifferent in the Sequence Listing.

FIG. 43 shows nucleotide sequence of the Hu119 VL gene flanked by NheIand EcoRI sites (underlined) is shown along with the deduced amino acidsequence. Amino acid residues are shown in single letter code. Thesignal peptide sequence is in italic. The N-terminal amino acid residue(E) of the mature VL is double-underlined. CDR sequences according tothe definition of Kabat et al. (1991) are underlined. The intronsequence is in italic. Hu119 VL gene fragment digested with NheI andEcoRI was cloned between the corresponding sites in the ExpressionVector shown in FIG. 23. The same sequences as claimed herein are alsoprovided in the Sequence Listing although the position numbers may bedifferent in the Sequence Listing.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., hOX40). It has been shown thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH-1 domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. Other forms of single chain antibodies, such asdiabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123). Still further, an antibody or antigen-bindingportion thereof may be part of a larger immunoadhesion molecules, formedby covalent or non-covalent association of the antibody or antibodyportion with one or more other proteins or peptides. Examples of suchimmunoadhesion molecules include use of the streptavidin core region tomake a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein. Preferred antigen binding portions are completedomains or pairs of complete domains.

OX40/OX40-ligand (OX40 Receptor)/(OX40L) are a pair of costimulatorymolecules critical for T cell proliferation, survival, cytokineproduction, and memory cell generation. Early in vitro experimentsdemonstrated that signaling through OX40 on CD4⁺ T cells lead to TH2,but not TH1 development. These results were supported by in vivo studiesshowing that blocking OX40/OX40L interaction prevented the induction andmaintenance of TH2-mediated allergic immune responses. However, blockingOX40/OX40L interaction ameliorates or prevents TH1-mediated diseases.Furthermore, administration of soluble OX40L or gene transfer of OX40Linto tumors were shown to strongly enhance anti-tumor immunity in mice.Recent studies also suggest that OX40/OX40L may play a role in promotingCD8 T cell-mediated immune responses. As discussed herein, OX40signaling blocks the inhibitory function of CD4⁺CD25⁺ naturallyoccurring regulatory T cells and the OX40/OX40L pair plays a criticalrole in the global regulation of peripheral immunity versus tolerance.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (seee.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(See Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242).

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hOX40 is substantially free ofantibodies that specifically bind antigens other than hOX40). Anisolated antibody that specifically binds hOX40 may bind OX40 moleculesfrom other species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-human OX40 antibody that binds to an OX40 antigen and/or theactivation potency of an antibody, for example, an anti-OX40 antibodywhose binding to hOX40 receptor activates the biological activity ofhOX40 or activation of receptor binding in a human L/OX40 cell assay.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex. The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The phrase “surface plasmon resonance” includes an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore system (Pharmacia Biosensor AB,Uppsala, Sweden and Piscataway, N.J.). For further descriptions, seeExample 5 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26;Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., etal. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991)Anal. Biochem. 198:268-277.

The term “vector” includes a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “monoclonal antibody” (monoclonal antibody) refers to anantibody, or population of like antibodies, obtained from a populationof substantially homogeneous antibodies, and is not to be construed asrequiring production of the antibody by any particular method, includingbut not limited to, monoclonal antibodies can be made by the hybridomamethod first described by Kohler and Milstein (Nature, 256: 495-497,1975), or by recombinant DNA methods.

The term “chimeric antibody” (or “chimeric immunoglobulin”) refers to amolecule comprising a heavy and/or light chain which is identical withor homologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (Cabilly et al. (1984), infra; Morrison et al.,Proc. Natl. Acad. Sci. U.S.A. 81:6851).

The term “humanized antibody” refers to forms of antibodies that containsequences from non-human (eg, murine) antibodies as well as humanantibodies. A humanized antibody can include conservative amino acidsubstitutions or non-natural residues from the same or different speciesthat do not significantly alter its binding and/or biologic activity.Such antibodies are chimeric antibodies that contain minimal sequencederived from non-human immunoglobulins. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary-determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat, camel, bovine, goat, or rabbit having thedesired properties. Furthermore, humanized antibodies can compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences. These modifications are made tofurther refine and maximize antibody performance. Thus, in general, ahumanized antibody will comprise all or substantially all of at leastone, and in one aspect two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), or that of a human immunoglobulin (see, e.g.,Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E.A. et al., European Patent Application No. 0,519,596 A1; Queen et al.(1989) Proc. Natl. Acad. Sci. USA, Vol 86:10029-10033).

Each of the antibodies described and claimed herein may be referred to,in the singular or plural, as: “anti-OX40 antibody;” “anti-hOX40antibody;” “anti-hOX40 monoclonal antibody;” “anti-hu man OX40antibody;” “anti-human OX40 mAb;” “anti-hOX40 mAb” “hOX40 specificmonoclonal antibody;” “anti-OX40L antibody;” “anti-hOX40L antibody;”“anti-human OX40L antibody;” “human OX40 specific antibody;” “human OX40specific monoclonal antibody;” “human OX40 specific antibody;”“anti-human OX40 specific antibody;” “anti-human OX40 specificmonoclonal antibody;” “h-OX40 specific antibody;” “h-OX40 specificmonoclonal antibody;” “hOX40 agonistic antibody;” “hOX40 antagonist”and/or other similar variations of the same.

As disclosed in U.S. patent application Ser. No. 11/659,266 titled“Methods to Treat Disease States by Influencing the Signaling ofOX-40-Receptors and High Throughput Screening Methods and IdentifyingSubstrates Thereof” which is herein incorporated by reference, it wasdiscovered that a function of OX40L is the negative regulation of thegeneration of Tr1 cells induced by immunosuppressive agents Dex and vitD3, ICOSL, or immature DCs. This discovery demonstrates a generalmechanism by which OX40L enhances immunity and breaks immunologicaltolerance.

With the use of immunohistologic analysis (FIG. 1), intracellularstaining (FIG. 2), and cell sorting (FIG. 3), we have shown that bothICOS⁺IL-10-producing and ICOS-TGF-β-producing Tregs infiltrated human FLtissues. These FL-derived FOXP3⁺ Tregs can strongly inhibit theproliferation of FOXP3⁻CD4⁺CD25⁻ tumor-infiltrating T cells in responseto CD40⁻ ligand preactivated autologous lymphoma cells (FIG. 4). Thesuppressive activity of ICOS⁺ Tregs could be partially blocked by aneutralizing anti-IL-10 antibody, confirming the role of ICOS⁺IL-10producing Tregs in FL (FIG. 4). In the experiment of FIG. 2, tumor cellsand PBMCS were obtained from 7 patients with an initial diagnosis beforetherapy. PBMC's were also obtained from 7 healthy donors for comparison.The percentages of regulatory T cells over total CD4⁺ T cells weredetermined by flow cytometric analysis of CD4⁺CD25⁺CD127^(low)FOXP3⁺Tregs. FIG. 2A provides representative FACS analysis of Tregs, whileFIG. 2B shows the percentage of Tregs of all donors.

It was also discovered that OX40L inhibits the generation of Tr1 cellsfrom CD4⁺ T cells induced by Dex and vit D3. It is known that acombination of the immunosuppressive drugs Dex and vit D3 consistentlyinduce the differentiation of naïve CD4⁺ T cells into Tr1 cells. Toinvestigate whether OX40L can inhibit the generation and function of Tr1cells, naïve CD4⁺ T cells were cultured with anti-CD3 plus anti-CD28monoclonal antibodies in the presence or absence of OX40L-transfected Lcells in four different culture conditions including: (1) Tr1 (Dex andvit D3); (2) TH1 (IL-12); (3) TH2 (IL-4); or (4) neutral (medium alone)for 7 days (FIG. 5A). IL-10 production by the primed T cells wasanalyzed by intracellular cytokine staining and ELISA.

In the experiments of FIG. 5A, an intracellular analysis of cytokineproduction by naïve CD4⁺ T cells was conducted by flow cytometry. NaïveCD4⁺ T cells were cultured with anti-CD3 and anti-CD28 monoclonalantibodies in the presence of IL-2 on parental L cells or OX40L-L cellswith the indicated recombinant cytokines or reagents for 7 days.Percentages of the respective cytokine-producing T cells are indicatedin each dot blot profile. The results show that OX40L inhibits thegeneration of Tr1 cells from naïve CD4⁺ T cells induced by the differentpolarizing signals. As shown in FIG. 5A, between 2% to 4% of Tr1 cellswere generated from naïve CD4⁺ T cells cultured in neutral or TH1 or TH2conditions. More than 15% of Tr1 cells were generated in culture withDex plus vit D3. The addition of OX40L completely blocked the generationof Tr 1 cells, while promoting the generation of TNF-α-producing T cellsin all culture conditions.

These data were confirmed by ELISA data (FIG. 5B). In the experiments ofFIG. 5B, cytokine production by naïve CD4⁺ cells in supernatants afterrestimulation with anti-CD3 and anti-CD28 monoclonal antibodies for 24 hwas measured by ELISA. Naïve CD4⁺ T cells were cultured with anti-CD3and anti-CD28 monoclonal antibodies in the presence of IL-2 on parentalL cells or OX40L-L cells with the indicated recombinant cytokines orreagents for 7 days. The data are shown as mean±standard error of themean (SEM) of four independent experiments. The results show that OX40Linhibits the generation of Tr1 cells from naïve CD4⁺ T cells induced bythe different polarizing signals.

Naïve CD4⁺ T cells primed with Tr1 condition (Dex plus vit D3) wereanergic and had the ability to suppress the proliferation of naïve CD4⁺T cells in response to anti-CD3 plus anti-CD28 monoclonal antibodies(FIG. 5C). In the experiments of FIG. 5C, suppressive function in Tcells was measured by [3-H]thymidine incorporation. Mixtures of theindicated T cell populations were restimulated by anti-CD3 and anti-CD28monoclonal antibodies. Error bars represent SEM of triplicate wells. Itwas discovered that naïve CD4⁺ T cells primed with the same Tr1condition in the presence of OX40L proliferated vigorously and failed toinhibit the proliferation of naïve CD4⁺ T cells in response to anti-CD3plus anti-CD28 monoclonal antibodies. The data suggest that OX40L blocksthe generation of functional Tr1 cells from naïve CD4⁺ T cells inducedby Dex and vit D3.

It was discovered that Tr1 cells can be generated from memoryCD4⁺CD45RA⁻ CD45RO⁺ T cells, and that OX40L can inhibit the generationof Tr1 cells from memory CD4⁺ T cells. Memory CD4⁺CD45RA⁻CD45RO⁺ T cellswere cultured for 7 days with anti-CD3 plus anti-CD28 monoclonalantibodies in the presence or absence of OX40L-transfected L cells Tr1condition (Dex plus vit D₃). In the experiments of FIG. 6A, anintracellular analysis of cytokine production by CD4⁺ memory T cells wasconducted by flow cytometry. Memory CD4⁺CD45RO⁺CD25⁻ memory T cells werecultured with anti-CD3, anti-CD28 monoclonal antibodies, and IL-2 onparental L cells or OX40L-L cells in the presence or absence of Dex plusvit D3 for 7 days. Percentages of the respective cytokine-producing Tcells are indicated in each dot blot profile. The results show thatOX40L inhibits the generation of Tr1 cells from memory CD4⁺ T cellsunder a condition with Dex plus vit D3. FIG. 6A shows that large numbersof Tr1 cells (>20%) were generated from CD4⁺ memory T cells in culturewith Dex plus vit D3. The addition of OX40L completely blocked thegeneration of Tr1 cells and promoted generation of TNF-α-producing cellsfrom memory CD4⁺ T cells.

The ability of Dex plus vit D3 to promote IL-10 production from memoryCD4⁺ T cells, and that this ability can be inhibited by OX40L, wereconfirmed by IL-10 ELISA analyses (FIG. 6B). In the experiments of FIG.6B, IL-10 production by memory CD4⁺ T cells was measured in supernatantsafter restimulation with anti-CD3 and anti-CD28 monoclonal antibodiesfor 24 h by ELISA. The data are shown as mean±SEM of four independentexperiments. The results show that OX40L inhibits the generation of Tr1cells from memory CD4⁺ T cells under a condition with Dex plus vit D3.

It was further discovered that OX40L inhibits the generation of Tr1cells, while other TNF-family members (GITRL and 4-1BBL) do not. Withinthe TNF-superfamily, OX40L, glucocorticoid-induced TNF receptor-ligand(GITRL), and 4-1BB-ligand (4-1BBL) have costimulatory function for Tcells. To investigate whether OX40L was unique in the inhibition of Tr1cells, naïve (CD4⁺ T cells were cultured with anti-CD3 plus anti-CD28monoclonal antibodies with Dex plus vit D3, with parental L cells or Lcells transfected with OX40L, GITRL, or 4-1 BBL for 7 days. While OX40L,GITRL, and 4-1BBL all promoted the generation of TNF-α-producing cells,only OX40L inhibited the generation of Tr 1 cells (FIGS. 7A and 7B).

In the experiments of FIG. 7A, an intracellular analysis of cytokineproduction by naïve CD4⁺ T cells was conducted by flow cytometry. NaïveCD4⁺ T cells were cultured with anti-CD3, anti-CD28 monoclonalantibodies, and IL-2 on parental L cells, OX40L-L cells, GITRL-L cells,or 4-1BBL-L cells in the presence of Dex plus vit D3 for 7 days.Percentages of the respective cytokine-producing T cells are indicatedin each dot blot profile. The results show that OX40L but not GITRL nor4-1BBL inhibits the generation of Tr1 cells.

In the experiments of FIG. 7B, IL-10 by naïve CD4⁺ cells was measured insupernatants after restimulation with anti-CD3 and anti-CD28 monoclonalantibodies for 24 h by ELISA. The data are shown as mean±SEM of fourindependent experiments. The results show that OX40L but not GITRL nor4-1BBL inhibits the generation of Tr1 cells.

OX40L, GITRL, and 4-1BBL all promoted the expansion of total T cellnumbers (FIG. 7C). In the experiments of FIG. 7C, the number of viable Tcells was counted. The data are shown as mean±SEM of four independentexperiments.

As understood by those of skill in the art, the results of FIGS. 7A, 7B,and 7C show that OX40L, but not GITRL nor 4-1BBL, inhibits thegeneration of Tr1 cells. These data suggest that among the three membersof TNF-superfamily known to costimulate T cells, OX40L has a novel andunique function in inhibiting the generation of Tr1 cells.

It was further discovered that OX40L inhibits the generation of Tr1cells induced by ICOSL or immature DCs. ICOS and CD28 represent the twopositive costimulatory receptors within the CD28 family expressed on Tcells. Signaling through ICOS by agonistic antibodies or ICOSL has beenshown to promote CD4⁺ T cells to produce IL-10. To investigate whetherOX40L can inhibit the ability of ICOS to induce IL-10 production by CD4⁺T cells, naïve and memory CD4⁺ T cells were cultured with anti-CD3 inthe presence of ICOSL-transfected L cells, or ICOSL-transfected L cellsin the presence of OX40L for 7 days.

In the experiments of FIG. 8A, an intracellular analysis of cytokineproduction by naïve CD4⁺ T cells was conducted by flow cytometry. NaïveCD4⁺ T cells were cultured for 7 days on parental L cells, on a mixtureof ICOSL-L cells and L cells, or on a mixture of ICOSL-L cells andOX40L-L cells, which were pre-coated with anti-CD3 monoclonal antibody.Percentages of the respective cytokine-producing T cells are indicatedin each dot blot profile. The results show that OX40L inhibits thegeneration of Tr1 cells from naïve CD4⁺ T cells induced by ICOSL.

In the experiments of FIG. 8B, IL-10 production by naïve CD4⁺ cells wasmeasured in supernatants after restimulation with anti-CD3 and anti-CD28monoclonal antibodies for 24 h was measured by ELISA. Naïve CD4⁺ T cellswere cultured for 7 days on parental L cells, on a mixture of ICOSL-Lcells and L cells, or on a mixture of ICOSL-L cells and OX40L-L cells,which were pre-coated with anti-CD3 monoclonal antibody. The data areshown as mean±SEM of three independent experiments. The results showthat OX40L inhibits the generation of Tr1 cells from naïve CD4⁺ T cellsinduced by ICOSL.

In the experiments of FIG. 8C, an intracellular analysis of cytokineproduction by memory CD4⁺ T cells was conducted by flow cytometry.Memory CD4⁺ T cells were cultured for 7 days on parental L cells, on amixture of ICOSL-L cells and L cells, or on a mixture of ICOSL-L cellsand OX40L-L cells, which were pre-coated with anti-CD3 monoclonalantibody. Percentages of the respective cytokine-producing T cells areindicated in each dot blot profile. The results show that OX40L inhibitsthe generation of Tr1 cells from memory CD4⁺ T cells induced by ICOSL.

In the experiments of FIG. 8D, IL-10 production by memory CD4⁺ T cellsin supernatants after restimulation with anti-CD3 and anti-CD28monoclonal antibodies for 24 h was measured by ELISA. Memory CD4⁺ Tcells were cultured for 7 days on parental L cells, on a mixture ofICOSL-L cells and L cells, or on a mixture of ICOSL-L cells and OX40L-Lcells, which were pre-coated with anti-CD3 monoclonal antibody. The dataare shown as mean±SEM of three independent experiments. The results showthat OX40L inhibits the generation of Tr1 cells from memory CD4⁺ T cellsinduced by ICOSL.

The results of the experiments of FIGS. 8A, 8B, 8C, and 8D show thatICOSL significantly promoted the generation of Tr1 cells from both naïveand memory CD4⁺ T cells. The addition of OX40L completely inhibited thegeneration of Tr1 cells from both naïve and memory CD4⁺ T cells, whilestrongly promoting the generation of cells producing TNF-α.

It is known that immature DCs or DCs treated with IFN-α or IL-10) caninduce naïve CD4⁺ T cells to differentiate into Tr1 cells. It wasinvestigated whether OX40L could inhibit the generation of Tr1 cellsinduced by DCs. As shown in FIG. 8E, immature DCs or DCs treated withIL-10 or IFN-α all induced the generation of more than 10% of Tr1 cellsfrom naïve CD4⁺ T cells. By contrast, DCs activated by CD40L induce astrong TH1 response, accompanied by the generation of about 3% Tr1cells. Addition of recombinant OX40L in DC-T cell cultures completelyinhibited the generation of Tr1 cells induced by immature DCs and DCstreated with IL-10 and IFN-α. In addition, OX40L also inhibited thegeneration of the residual number of Tr1 cells induced by the CD40Lactivated mature DCs. In the experiments of FIG. 5E, an intracellularanalysis of cytokine production by CD4⁺ naïve T cells was conducted byflow cytometry. Naïve CD4⁺ T cells were cocultured in the presence orabsence of soluble recombinant OX40L for 7 days with immature DCs or DCscultured with IFN-α, IL-10, and CD40L. Percentages of the respectivecytokine-producing T cells are indicated in each dot blot profile. Theresults show that OX40L inhibits the generation of Tr1 cells from CD4⁺ Tcells induced by DCs

The ability of OX40L to inhibit the generation of Tr1 cells induced byDCs was confirmed by ELISA data (FIG. 8F). In the experiments of FIG.8F, IL-10 production by naïve CD4⁺ cells was measured in supernatantsafter restimulation with anti-CD3 and anti-CD28 monoclonal antibodiesfor 24 h by ELISA. Naïve CD4⁺ T cells were cocultured in the presence orabsence of soluble recombinant OX40L for 7 days with immature DCs or DCscultured with IFN-α, IL-10, and CD40L. The data are shown as mean±SEM ofthree independent experiments. The results show that OX40L inhibits thegeneration of Tr1 cells from CD4⁺ T cells induced by DCs. Thus, thesedata demonstrate that OX40L could inhibit the generation of Tr1 cellsinduced by more physiological signals provided by ICOSL and DCs.

It has been previously suggested that regulatory T cells are highlyrepresented in the area of B cell non-Hodgkin's lymphoma and that Bcells are involved in the recruitment of regulatory T cells into thearea of the lymphoma. It was investigated whether influencing thesignaling of OX40-receptors, such as by OX40L, could provide a therapyagainst B cell lymphoma. Cryopreserved samples from B cell lymphomapatients were used to estimate the ability of OX40L to shut down Tr1cells. The samples used were follicular lymphoma obtained from a spleenspecimen prior to any treatment. The cells were thawed, with 400×10⁶frozen cells yielding 127×10⁶ live cells and 33.9×10 dead cells (79%viability). A sufficient number of CD25⁺ cells were identified by FACSstaining. In the experiments of FIG. 9, IL-10 secretion of ICOS⁺IL-10producing Tregs was determined by ELISA. Treg cells were cultured undertwo different conditions. In condition 1, CD25⁺/ICOS⁺ cells werecultured with anti-CD3 in the presence of IL-2 (900 μl/ml) on parental Lcells or OX40L-L cells with anti-ICOS antibody for 3-6 days. Incondition 2, CD25⁺/ICOS⁺ cells were cultured with anti-CD3 in thepresence of IL-2 (900 μl/ml) on ICOS-L-L cells or a mixture of OX40L-Lcan ICOS-L-L cells for 3 to 6 days. Cytokine production in thesupernatants was measured by ELISA. The results show that OX40L greatlyinhibited IL-10 production by Treg cells.

The findings, that OX40L has the capacity to inhibit the generation andfunction of Tr1 cells induced by the immunosuppressive drugs Dex plusvit D3, ICOSL, or DCs, highlights a novel mechanism by which OX40Lpromotes immunity and breaks tolerance during different forms of CD4- orCD8-mediated immune responses, as would be understood by one of skill inthe art. The ability of OX40L to inhibit the generation of Tr1 cellsduring both IL-12 induced TH1 or IL-4 induced TH2 responses suggest thatOX40L may control the magnitude of TH1- or TH2-mediated immuneresponses. Furthermore, the ability of OX40L to inhibit the generationof Tr1 cells appears to be a unique property of OX40L, because the twoother TNF-family members GITRL and 4-1BBL do not have this functionalproperty. Moreover, the ability of OX40L to inhibit IL-10 production byTreg cells identifies OX40L as a potent treatment for B cell lymphomaand other cancers.

Many molecules have been identified that promote the generation of Tr1cells, including IL-10, IFN-α, ICOSL, and immunosuppressive compoundssuch as Dex plus vit D3. OX40L represents a potent inhibitor for thegeneration of Tr1 cells not only from naïve CD4⁺ T cells, but also frommemory CD4⁺ T cells and regulatory T cells. This novel property ofOX40/OX40L may explain a recent report showing that OX40 signalingallows anergic autoreactive T cells to acquire effector cell functions.Targeting OX40/OX40L thus provides for treatments for human allergic andautoimmnune diseases and as well as for the development of treatmentsfor human infectious diseases and cancer including but not limited tomelanoma, brain cancer, bone cancer, a leukemia, a lymphoma, epithelialcell-derived neoplasia (epithelial carcinoma) such as basal cellcarcinoma, adenocarcinoma, gastrointestinal cancer such as lip cancer,mouth cancer, esophageal cancer, small bowel cancer and stomach cancer,colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarycancer, cervical cancer, lung cancer, breast cancer and skin cancer,such as squamous cell and basal cell cancers, prostate cancer, renalcell carcinoma, and other known cancers.

Disorders or conditions that can be prevented or treated by antibodiesand methods described herein include the prevention or treatment ofcancer, such as cutaneous T-cell leukemia, head and neck tumors,pancreatic cancer, bladder cancer, high grade gliomas, brain metastasis,melanoma, skin cancer, lung cancer, breast cancer, prostate cancer,colon cancer, leukemia, myelodysplastic syndrome (a pre-leukemiacondition), and multiple myeloma. In general, metastasis of any cancercan be prevented or treated with the compounds and methods describedherein. The antibodies may also be used to prevent or treatproliferative angiogenic conditions including telangectasia, venousangiomas, hemangioblastoma. Other disorders, diseases or conditionsinclude viral diseases, some of which may traditionally considered“untreatable.” The antibodies, for example, may also be used to classifystrains of a single pathogen. Researchers can use the antibodiesdescribed herein to identify and to trace specific cells or molecules inan organism.

Generally, the terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. More specifically, cancers which can be treatedor prevented using any one or more of the antibodies described herein ora variant thereof, include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include, but are not limited to, squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer and gastrointestinal stromal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma and various types of head andneck cancer, melanoma, superficial spreading melanoma, lentigo malignamelanoma, acral lentiginous melanomas, nodular melanomas, as well asB-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NIL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

Methods for treating or preventing an immune disorder are also providedherein. These methods comprising administering an effective amount ofthe antibody to a subject in need of such treatment. In someembodiments, the immune disorder is an immune disorder or an autoimmunedisorder. The disorder is asthma, atopic dermatitis, allergic rhinitis,inflammatory bowel disease, multiple sclerosis, GVHD, and/or systemiclupus erythematosus. In some embodiments, the disorder is a diseaseassociated with virus, bacteria or other infectious agent.

Moreover, the antibodies and methods that are described herein can beused to prevent or treat inflammatory diseases and conditions, such asosteoarthritis, Rheumatoid arthritis, Crohn's disease, ulcerativecolitis, and auto-immune diseases such as lupus and mixed auto-immunedisease. For example, the antibodies described herein may be useful intreating a variety of autoimmune and inflammatory disease comprising thestep of administering a therapeutically effective amount of the antibodyto a subject in need thereof, wherein the autoimmune disease orinflammatory disease is any one or more of the following diseases:insulin-dependent diabetes mellitus (IDDM), diabetes mellitus, multiplesclerosis, experimental autoimmune encephalomyelitis, acute disseminatedencephalomyelitis, arthritis, rheumatoid arthritis, experimentalautoimmune arthritis, myasthenia gravis, thyroiditis, Hashimoto'sdisease, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmuneatrophic gastritis, Addison's disease, premature menopause, maleinfertility, juvenile diabetes, Goodpasture's syndrome, pemphigusvulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis,autoimmune haemolyticanaemia, idiopathic leucophenia, primary biliarycirrhosis, active chronic hepatitis Hb_(s-ve), cryptogenic cirrhosis,ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener'sgranulomatosis, Poly/Dermatomyositis, discoid LE, systemic Lupuserythematosus, Chron's disease, psoriasis, Ankylosingspondylitisis,Antiphospholipid antibody syndrome, Aplastic anemia, Autoimmunehepatitis, Coeliac disease, Graves' disease, Guillain-Barre syndrome(GBS), Idiopathic thrombocytopenic purpura, Opsoclonus myoclonussyndrome (OMS), Optic neuritis, ORd's thyroiditis, Pemphigus,Polyarthritis, Primary biliary cirrhosis, Reiter's syndrome, Takayasu's,Temporal arteritis, Warm autoimmune hemolytic anemia, Wegener'sgranulomatosis, Alopecia universalis, Behcet's disease, Chagas' disease,Chronic fatigue syndrome, Dysautonomia, Endometriosis, Hidradenitissuppurativa, Interstitial cystitis, Neuromyotonia, Sarcoidosis,Scleroderma, Ulcerative colitis, Vitiligo, Vulvodynia, inflammatory skindiseases, allergic contact dermatitis, H. pylory gastritis, chronicnasal inflammatory disease, arteriosclerosis and graft versus hostdisease.

More specifically, an “autoimmune disease” as referred herein is adisease or disorder arising from and directed against an individual'sown tissues or organs or a co-segregate or manifestation thereof orresulting condition there from. Autoimmune disease may refer to acondition that results from, or is aggravated by, the production by Bcells of antibodies that are reactive with normal body tissues andantigens. Also, an autoimmune disease is one that may involve thesecretion of an autoantibody that is specific for an epitope from a selfantigen (e.g. a nuclear antigen).

Autoimmune diseases or disorders that are treatable and/or preventableby any one or more of the antibodies described herein include, but arenot limited to, arthritis (rheumatoid arthritis such as acute arthritis,chronic rheumatoid arthritis, gout or gouty arthritis, acute goutyarthritis, acute immunological arthritis, chronic inflammatoryarthritis, degenerative arthritis, type II collagen-induced arthritis,infectious arthritis, Lyme arthritis, proliferative arthritis, psoriaticarthritis, Still's disease, vertebral arthritis, and juvenile-onsetrheumatoid arthritis, osteoarthritis, arthritis chronica progrediente,arthritis deformans, polyarthritis chronica primaria, reactivearthritis, and ankylosing spondylitis), inflammatory hyperproliferativeskin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis,pustular psoriasis, and psoriasis of the nails, atopy including atopicdiseases such as hay fever and Job's syndrome, dermatitis includingcontact dermatitis, chronic contact dermatitis, exfoliative dermatitis,allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, nummular dermatitis, seborrheic dermatitis, non-specificdermatitis, primary irritant contact dermatitis, and atopic dermatitis,x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria, myositis,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO),inflammatory bowel disease (IBD) (for example, Crohn's disease,autoimmune-mediated gastrointestinal diseases, colitis such asulcerative colitis, colitis ulcerosa, microscopic colitis, collagenouscolitis, colitis polyposa, necrotizing enterocolitis, and transmuralcolitis, and autoimmune inflammatory bowel disease), bowel inflammation,pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis,respiratory distress syndrome, including adult or acute respiratorydistress syndrome (ARDS), meningitis, inflammation of all or part of theuvea, iritis, choroiditis, an autoimmune hematological disorder,rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema,cranial nerve damage as in meningitis, herpes gestationis, pemphigoidgestationis, pruritis scroti, autoimmune premature ovarian failure,sudden hearing loss due to an autoimmune condition, IgE-mediateddiseases such as anaphylaxis and allergic and atopic rhinitis,encephalitis such as Rasmussen's encephalitis and limbic and/orbrainstem encephalitis, uveitis, such as anterior uveitis, acuteanterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive UN,proliferative nephritis, autoimmune polyglandular endocrine failure,balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, allergic reaction, eczema including allergic or atopiceczema, asteatotic eczema, dyshidrotic eczema, and vesicularpalmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma,and auto-immune asthma, conditions involving infiltration of T cells andchronic inflammatory responses, immune reactions against foreignantigens such as fetal A-B-O blood groups during pregnancy, chronicpulmonary inflammatory disease, autoimmune nocarditis, leukocyteadhesion deficiency, lupus, including lupus nephritis, lupus cerebritis,pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus anddiscoid lupus erythematosus, alopecia lupus, systemic lupuserythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatricinsulin-dependent diabetes mellitus (IDDM), adult onset diabetesmellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetesinsipidus, diabetic retinopathy, diabetic nephropathy, diabeticlarge-artery disorder, immune responses associated with acute anddelayed hypersensitivity mediated by cytokines and T-lymphocytes,tuberculosis, sarcoidosis, granulomatosis including lymphomatoidgranulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides,including vasculitis, large-vessel vasculitis (including polymyalgiarheumatica and giant-cell (Takayasu's) arteritis), medium-vesselvasculitis (including Kawasaki's disease and polyarteritisnodosa/periarteritis nodosa), microscopic polyarteritis,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as systemic necrotizingvasculitis, and ANCA-associated vasculitis, such as Churg-Straussvasculitis or syndrome (CSS) and ANCA-associated small-vesselvasculitis, temporal arteritis, aplastic anemia, autoimmune aplasticanemia, Coombs positive anemia, Diamond Blackfan anemia, hemolyticanemia or immune hemolytic anemia including autoimmune hemolytic anemia(AIHA), pernicious anemia (anemia perniciosa), Addison's disease, purered cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease,Parkinson's disease, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Behcet'sdisease/syndrome, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, thermal injury,preeclampsia, an immune complex disorder such as immune complexnephritis, antibody-mediated nephritis, polyneuropathies, chronicneuropathy such as IgM polyneuropathies or IgM-mediated neuropathy,thrombocytopenia (as developed by myocardial infarction patients, forexample), including thrombotic thrombocytopenic purpura (TTP),post-transfusion purpura (PTP), heparin-induced thrombocytopenia, andautoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, polyglandular syndromes such asautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis(LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barresyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, acute febrile neutrophilicdermatosis, subcorneal pustular dermatosis, transient acantholyticdermatosis, cirrhosis such as primary biliary cirrhosis andpneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliacdisease, celiac sprue (gluten enteropathy), refractory sprue, idiopathicsprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; LouGehrig's disease), coronary artery disease, autoimmune ear disease suchas autoimmune inner ear disease (AIED), autoimmune hearing loss,polychondritis such as refractory or relapsed or relapsingpolychondritis, pulmonary alveolar proteinosis, Cogan'ssyndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet'sdisease/syndrome, rosacea autoimmune, zoster-associated pain,amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis,which includes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal or segmental or focal segmentalglomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis,chorioretinitis, autoimmune hepatological disorder, fibromyalgia,multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastricatrophy, presenile dementia, demyelinating diseases such as autoimmunedemyelinating diseases and chronic inflammatory demyelinatingpolyneuropathy, Dressler's syndrome, alopecia greata, alopecia totalis,CREST syndrome (calcinosis, Raynaud's phenomenon, esophagealdysmotility, sclerodactyly, and telangiectasia), male and femaleautoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixedconnective tissue disease, Chagas' disease, rheumatic fever, recurrentabortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitissuch as allergic alveolitis and fibrosing alveolitis, interstitial lungdisease, transfusion reaction, leprosy, malaria, parasitic diseases suchas leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathicpulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatumet diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, flariasis, cyclitis such as chroniccyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), orFuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus(HIV) infection, SCID, acquired immune deficiency syndrome (AIDS),echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis,parvovirus infection, rubella virus infection, post-vaccinationsyndromes, congenital rubella infection, Epstein-Barr virus infection,mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronichypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemickeratoconjunctivitis, idiopathic nephritic syndrome, minimal changenephropathy, benign familial and ischemia-reperfusion injury, transplantorgan reperfusion, retinal autoimmunity, joint flammation, bronchitis,chronic obstructive airway/pulmonary disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, asperniogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning,conditions involving infiltration of T cells, leukocyte-adhesiondeficiency, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, diseasesinvolving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, allergic neuritis, autoimmune polyendocrinopathies,oophoritis, primary myxedema, autoimmune atrophic gastritis, sympatheticophthalmia, rheumatic diseases, mixed connective tissue disease,nephrotic syndrome, insulitis, polyendocrine failure, autoimmunepolyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism(AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisisbullosa acquisita (EBA), hemochromatosis, myocarditis, nephroticsyndrome, primary sclerosing cholangitis, purulent or nonpurulentsinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, orsphenoid sinusitis, an eosinophil-related disorder such as eosinophilia,pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonaryeosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing cosinophils, anaphylaxis, seronegativespondyloarthritides, polyendocrine autoimmune disease, sclerosingcholangitis, sclera, episclera, chronic mucocutaneous candidiasis,Bruton's syndrome, transient hypogammaglobulinemia of infancy,Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis,autoimmune disorders associated with collagen disease, rheumatism,neurological disease, lymphadenitis, reduction in blood pressureresponse, vascular dysfunction, tissue injury, cardiovascular ischemia,hyperalgesia, renal ischemia, cerebral ischemia, and diseaseaccompanying vascularization, allergic hypersensitivity disorders,glomerulonephritides, reperfusion injury, ischemic re-perfusiondisorder, reperfusion injury of myocardial or other tissues,lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses withacute inflammatory components, multiple organ failure, bullous diseases,renal cortical necrosis, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

The antibodies described herein may have a variety of academic, medicaland commercial uses. The antibodies may be used in different types ofdiagnostic tests, for example, to detect a wide variety of diseases orthe presence of drugs (pharmaceuticals), toxins or other proteinsincluding hormones, either in vitro or in vivo. The antibodies describedherein may be useful in testing for disease, for example, in serum orblood of patients. The disease may including OX40 related diseases ordisease or indications not related to OX40 including various cancers,inflammatory or autoimmune disease. Antibodies may also be used in theradioimmuno-detection and radioimmuno-therapy of cancer, and some newtesting methods can utilize these described antibodies to target onlythe cell membranes of specific cell types, i.e., cancer.

The antibodies described herein could be made part of a kit or otherdiagnostic package. As such, provided herein is a diagnostic kit, orarticle of manufacture for use with the pretreatment method herein. Thediagnostic kit may comprise any one or more of the following:antagonist/antibody/drug reference material; positive controlneutralizing antibody (preferably goat of cyno monkey); Protein A+Gcolumn (e.g. Protein A/G column); delipidation reagent; immunoglobulinaffinity purification buffer(s) (for example binding, elution andneutralization buffers); complement serum; assay diluent for cells;instruction manual or literature; vial of frozen cells (for example,WIL2 cells); cell labeling reagent (such as CELL TITER GLO®), etc. Byway of example, the diagnostic kit may include but is not limited to:(a) delipidation reagent; (b) buffers (e.g. binding and elution buffers)for affinity purification of immunoglobulins; and (c) instruction manualinstructing the user of the diagnostic kit to use the kit to pre-treat abiological sample from an autoimmune disease or cancer subject prior toconducting a cell based bioassay (such as a neutralizing antibody assay)on the sample (e.g. to avoid the problem of serum interference). Thediagnostic kit optionally further comprises any one or more of: drugreference material, positive control neutralizing antibody, complementserum, assay diluent for cells, and cell labeling reagent, etc.

The antibodies and other discoveries described herein also provide forhigh throughput screening methods. More specifically, and as understoodby those skilled in the art, high throughput methods to screen forantagonistic or agonistic monoclonal antibodies or small molecules thatbind to OX40-receptors, and that can inhibit the generation and functionof Tr1 cells or promote the generation and function of Tr1 cells, aremade possible. In one such method, a human T cell line (SU-DHL-1) havingthe ability to produce IL-10 was transfected with the human OX40-gene(SUOX40). 100,000 SUOX40 cells were cultured with either 100,000 mousefibroblast cells (L cells) or 100,000 mouse fibroblast cells expressingthe human OX40-ligand (OX40-ligand L cells) in 96 well-plates. After 48hours of culture, culture supernatants were collected for themeasurement of IL-10 by IL-10-specific ELISA. In a representativeexperiment, 100,000 SUOX40 cells produced up to 6,000 pg/ml IL-10cultured in the absence of OX40-ligand. In the presence of OX40-ligand,100,000 SUOX40 cells produced less than 1,000 μg/ml IL-10. This culturemethod may be used to screen for, inter alia, antagonistic monoclonalantibodies or small molecules that block the ability of OX40-ligand toinhibit IL-10 production by SUOX40 cells. Alternatively, this culturemethod may be modified by replacing OX40-ligand expressing L cells withpotential agonistic monoclonal antibodies or small molecules specific toOX40 to determine, inter alia, their ability to inhibit IL-10 productionby SUOX40 cells.

The anti-OX40 antibodies described herein can be used as an assay or inan assay for testing or measuring the activity of a drug or othermolecule found in an organism or organic sample. They could also be usedin a quantitative assay to measure the amount of a substance in asample. Bioassays and immunoassays are among the many varieties ofspecialized biochemical assays by which these antibodies might be used.The anti-OX40 antibodies taught herein can be used in other assays tomeasure processes such as enzyme activity, antigen capture, stem cellactivity, and competitive protein binding.

Human GITRL, OX40L, 4-1BBL, ICOSL expressing L cells were generated byretroviral mediated transduction, as understood by those of skill in theart. Briefly, full-length coding sequence for human GITRL (Accession#NM_(—)005092), OX40L (Accession #NM_(—)003326), 4-1BBL (Accession#NM_(—)003811), ICOSL (Accession #NM_(—)015259) was amplified by RT-PCRwith RNA prepared from HSV-1 stimulated PBMCs. Subsequently the cDNAswere cloned into an MSCV based retroviral vector pMIGW2 and theresulting plasmids were verified by restriction enzyme digestion and DNAsequencing. To produce recombinant retrovirus, each vector wasco-transfected with packaging constructs pCL-gp (gag/pol) and pHCMV-VSVg(VSV glycoprotein envelop) in HEK293T cells. Two days later, the viruscontaining culture supernatants were harvested and used to infect CD32 Lcells at moi 100. Under this condition >95% cells were productivelytransduced.

Isolated CD14⁺ monocytes (purity >94%) were cultured in the presence of100 ng/ml GM-CSF and 50 ng/ml IL-4 (both from R&D) for 5 days, asunderstood by those of skill in the art. The resulting immature DCs werewashed and cultured for 24 h with IFN-α (1000 U/ml, PBL BiomedicalLaboratories), IL-10 (10 ng/ml, R&D), and irradiated CD40L-transfected Lcells (DC to L cell ratio, 4:1) to obtain mature DCs, as understood bythose of skill in the art.

Naïve CD4⁺ T cells and memory CD4⁺ T cells (each purity >99%) wereisolated from PBMCs using CD4⁺ T cell Isolation Kit II (Miltenyi Biotec)followed by cell sorting (CD4⁺CD45RA⁺CD45RO⁻CD25− fraction as naïve Tcells and CD4⁺CD45RA⁻CD45RO⁺CD25⁻ fraction as memory T cells), asunderstood by those of skill in the art. 4×10⁴ freshly purifiedallogeneic naïve CD4⁺ T cells were co-cultured with immature or culturedDCs (DC to T ratio, 1:10) in the presence or absence of recombinanthuman OX40L (R&D, 100 ng/ml) in round-bottomed 96-well culture platesfor 7 days, as understood by those of skill in the art. Purified CD4⁺ Tcells were also cultured with IL-12 (10 ng/ml, R&D), IL-4 (25 ng/ml,R&D), or combination of dexamethasone (5×10⁻⁸ M, Life Technologies) and1alpha,25-dihydroxyvitamin D3 (10⁻⁷ M) for 7 days in the presence ofsoluble anti-CD28 monoclonal antibody (CD28.2, 1 μg/ml) and IL-2 (50U/ml, R&D) on the irradiated CD32/OX40L-L cells, CD32/GITRL-L cells,CD32/4-1BBL-L cells, or parental CD32-L cells which had been pre-coatedwith anti-CD3 monoclonal antibody (OKT3, 0.2 μg/ml) in 48-well cultureplates (T cell to L cell ratio, 2.5:1), as understood by those of skillin the art. In some experiments, CD4⁺ T cells were cultured for 7 dayson the CD32-L cells, mixture of CD32-L cells and CD32/ICOSL-L cells(ratio 1:1), or mixture of CD32/ICOSL-L cells and CD32/OX40L-L cells(ratio 1:1) pre-coated with anti-CD3 monoclonal antibody (0.2 μg/ml) in48-well culture plates, as understood by those of skill in the art. RPMI1640 was used and supplemented with 10% FCS, 2 mM L-glutamine, 1 mMsodium pyruvate, penicillin G, and streptomycin for the cultures, asunderstood by those of skill in the art.

The cultured T cells were collected and washed, and then restimulatedwith plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2 μg/ml) at aconcentration of 1×10⁶ cells/ml for 24 h, as understood by those ofskill in the art. The levels of IL-4, IL-10, TNF-α, and IFN-α in thesupernatants were measured by ELISA (all kits from R&D), as understoodby those of skill in the art. For intracellular cytokine production, thecultured T cells were restimulated with 50 ng/ml of PMA plus 2 μg/ml ofionomycin for 6 h. Brefeldin A (10 μg/ml) was added during the last 2 h,as understood by those of skill in the art. The cells were stained witha combination of PE-labeled monoclonal antibodies to IL-4 or TNF-αFITC-labeled monoclonal antibodies to IFN-α and APC-labeled anti-IL-10(all from BD) using FIX and PERM kit (CALTAG), as understood by those ofskill in the art.

T cells were collected and re-suspended in an EDTA-containing medium todissociate the clusters, as understood by those of skill in the art.Viable cells were counted by trypan-blue exclusion of the dead cells, asunderstood by those of skill in the art. For suppressive function assay,naïve CD4⁺ T cells (A) and Tr1 cells generated from naïve CD4⁺ T cellsby anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, IL-2,Dex, and vit D3 in the presence of parental L cells (B) or OX40L-L cells(C), these three cell types and their mixtures at a 1:1 ratio were thenrestimulated for 5 days by culturing in the presence of 5 μg/ml anti-CD3monoclonal antibody and 1 μg/ml anti-CD28 monoclonal antibody, afterwhich time the cellular proliferation was assessed by [³H]thymidineincorporation, as understood by those of skill in the art.

Generation of Anti-Human OX40-Specific Monoclonal Antibodies

We generated multiple agonist mouse monoclonal antibodies against humanOX40. The antigen binding specificity of the antibodies was confirmed byflow cytometry (FIGS. 10-12). The agonist activity of the antibodies wasvalidated through functional assays. We found that nine of the 20OX40-specific antibodies could block vitamin D3/dexamethasone-mediatedgeneration of Tr1 cells from CD4⁺ T cells (FIG. 13), enhance CD4⁺ T-cellproliferation (FIG. 14), and suppress ICOS⁺CD4⁺CD25^(high)FOXP3⁺ TregIL-10 production (FIG. 16). We titrated the antibodies and found thatfive possessed potent activity in suppressing Tr1 cell generation atconcentrations as low as 4 ng/ml (FIG. 15).

OX40 Antibodies Inhibit CD4⁺CD25^(high)FOXP3⁺ Treg Function

Some of the OX40 monoclonal antibodies inhibit the suppressive functionof FOX P3⁺ Treg (FIG. 17). Of the five antibodies (119-8B, 119-43,119-122, 119-173B, and 106-222) that potently inhibit IL-10 productionfrom Tr1 cells and CD4⁺CD25^(high)CD127⁻FOXP3⁺ Tregs, three (119-43,119-122, and 106-222) were potent in blockingCD4⁺CD25^(high)CD127⁻FOXP3⁺ Treg function (FIG. 17). However, two(119-33 and 120-140A) of the 11 antibodies that have no activity againstIL-10 production, but block CD4⁺CD25^(high)FOXP3⁺ Treg function (FIG.18).

Anti-Human OX40 Monoclonal Antibodies

Generation of anti-human OX40 monoclonal antibodies was performed forexample, by immunizing 6-8-wk-old BALB/c mice with a mouse cell linetransfected with human-OX40 following established protocols. Hybridomaclones secreting monoclonal antibody that specifically stained OX40⁺cells were established and further analyzed.

We design an exhaustive screening to detect those clones that triggerOX40 signaling (i.e., agonists antibodies) by inhibiting the generationand function of Tr1 cells. Those clones were further purified. Agonistantibodies against hOX40 may be humanized and use in clinical protocolsfor human anti tumor therapy, either alone or in combination with antitumor vaccination and other adjuvants. Several different tumor typescould be the target of these antibodies, including melanoma, lymphomaand breast cancer.

In another embodiment, 6-8 week-old BALB/c female mice were used forfootpad or subcutaneous immunization. Each mouse was injected with 5million murine L cells transfected with human-OX40 (L-OX40) 6 times at 3days intervals. Three days after the sixth injection, mice weresacrificed and popliteal lymph nodes (from footpad immunization) orspleen (from subcut immunization) were removed and cells were fused withSP2.0 myeloma or NSO myeloma cells at a ratio of 1 to 1 to generatehybridoma clones using established protocols. Hybridoma clones secretingmonoclonal antibody were then screened for their binding specificity toL-hOX40 cells by ELISA assays. Hybridoma supernatants that bind toL-hOX40 cells and not L parental cells were further confirmed forbinding on L-hOX40 and SUPM2-hOX40 cells by flow cytometry analysis.

In the experiment of FIG. 10, hOX40 hybridoma supernatants were screenedagainst L-hOX40 versus L parental cells by ELISA. Twenty hOX40-specificmonoclonal antibodies were selected. Twenty million L cells or L cellsexpressing human OX40 (L-hOX40) were coated on a 96-well plate by mixingcells with 0.01% magnesium calcium chloride in PBS and let driedovernight in a laminar hood. Plates were then frozen at −20° C. for atleast one day before use. For antibody binding assays, frozen cells wererehydrated with PBS and washed with wash buffer containing PBS plus0.05% Twen 20, and blocked with 2% BSA in wash buffer. Conditioned cellswere then used for binding to OX40 antibody supernatants. Antibodybinding to cells was then detected with a secondary antibody, anti-mouseIgG FC HRP. hOX40-specific hybridoma supernatants recognize L cellexpressing OX40 but not parental L cells.

In the experiment of FIG. 11, hOX40-specific monoclonal antibodies werescreened by flow cytometry analysis. Equal number (100k) of L cells andL-hOX40 were mixed in FACS buffer (1% FCS/2 mM EDTA/PBS) and incubatedwith 0.5 μg of FPLC (Protein A HiTrap/Gentle Ag/Ab elution buffer)purified antibodies. Cells were then washed and stained with a secondaryantibody, PE-conjugated anti-mouse IgG. Two peaks indicate positive andnegative stain by anti-hOX40 monoclonal antibody. A single peak suggestsno binding or none-specific binding of antibodies. Twenty hOX40-specificmonoclonal antibodies were confirmed by flow cytometry analysis.

In the experiment of FIG. 12, hOX40 monoclonal antibodies specificitywas confirmed by using SUPM2 cells expressing hOX40 (SUPM2-hOX40). Equalnumber (100k) of SUPM2 and SUPM2-hOX40 cells were mixed in FACS buffer(1% FCS/2 mM EDTA/PBS) and used for hOX40 monoclonal antibody binding asin FIG. 11. The binding specificity of each antibody was analyzed byflow cytometry. Two peaks indicate positive and negative stain byanti-hOX40 monoclonal antibody, while a single peak suggests no bindingor none-specific binding by antibodies. Twenty hOX40-specific monoclonalantibodies were reconfirmed.

In the experiment of FIG. 13, we sought to identify human OX40-specificmonoclonal antibodies that can inhibit the generation of Tr1 cells fromCD4⁺ T cells stimulated by VitD₃ (10 microMole mM)/Dex (50 nanoM),CD32L/ICOSL and anti-CD3/CD28 (0.2 microgram/ml). Anti-hOX40 monoclonalantibodies were added on day 0 of cell culture and CD4⁺ T cells after 7days of stimulation were subjected to IL-10 intracellular stainingfollowed by flow cytometry analysis. Representative FluorescenceActivated Cell Sorting (FACS) data, are shown in A and the percentagesof Tr1 cells for all anti-hOX40 monoclonal antibodies treatments areshown in B. Using cells obtained from this experiment, we sought toidentify hOX40-specific monoclonal antibodies that stimulate CD4⁺ T cellproliferation (FIG. 14, cells were counted on day 7 after stimulation)and inhibit Tr1 generation from CD4⁺ (FIG. 13).

In order to identify such hOX40 monoclonal antibodies for their abilityto inhibit the generation of Tr1 cells from CD4⁺ T cells, Tr1 cells weregenerated and cultured as described in the experiments for FIG. 13above. Representative FACS data are shown in A and percentage of Tr1cells after treatment with nine anti-hOX40 monoclonal antibodies areshown in B. Five hOX40-specific monoclonal antibodies strongly inhibitedthe generation of Tr1 cells at 4 ng/ml concentration (FIG. 15).

In the experiment of FIGS. 16A, 16B, and 16C, freshly sortedICOS⁺CD4⁺CD127⁻ CD25^(high) T cells were stimulated with anti-CD3 (0.2μg/ml) in the presence of CD32L/ICOSL cells and CD32L/hOX40L cells oranti-hOX40 monoclonal antibodies or control antibody for 5 days. Then,cells were counted and 5×10⁴ cells were restimulated with anti-CD3/CD28for 24 hrs and supernatants were assayed for IL-10 secretion with anElisa kit. We identified hOX40-specific monoclonal antibodies thatinhibit Tr1 generation from CD4⁺ T cells also inhibit IL-10 productionfrom naturally ICOS⁺CD4⁺CD25^(high) T cells. Freshly sorted ICOS⁺ ICOS⁻CD4⁺CD127⁻CD25^(high) Tregs were cultured with CFSE-labeledCD4⁺CD25^(low) cells in the presence of irradiated monocytes andanti-CD3 (0.3 μg/ml) and anti-hOX40 mAbs. After 3.5 days of culture,cell proliferation was assessed for dilution of CFSE in cells by FACS(FIG. 16C).

FIGS. 17 A and 17B shows the identification of anti-hOX40 monoclonalantibodies that inhibit the generation of Tr1 cells and blockFOXP3⁺CD4⁺CD25^(high) Treg function. Freshly sortedFOXP3⁺CD4⁺CD127⁻CD25^(high) T cells (3.5×10⁴) were cultured withCFSE-labeled CD4⁺CD25^(low) cells (7×10⁴) in the presence of Irradiatedmonocytes (7×10⁴, 6000 rad) and 0.3 μg/ml anti-CD3 and variousconcentrations of anti-hOX40 monoclonal antibody. After 3 to 4 days ofculture, cell proliferation was assessed for dilution of CFSE in cellsby Flow cytometry analysis. Percentage of divided cells is indicated.Representative flow cytometry analyses are shown in FIG. 17A. Data, for6 monoclonal antibodies are shown in FIG. 17B.

In the experiment of FIG. 18, freshly sorted FOXP3⁺CD4⁺CD127⁻CD25^(high)T cells (3.5×10⁴) were cultured with CFSE-labeled CD4⁺CD25^(low) cells(7×10⁴) in the presence of irradiated monocytes (7×10⁴, 6000 rad) and0.3 μg/ml anti-CD3 and various concentrations of OX40 monoclonalantibody. After 3 to 4 days of culture, cell proliferation was assessedfor dilution of CFSE dye in cells by FACS. Data are representative oftwo experiments. We identified anti-hOX40 monoclonal antibodies that donot inhibit Tr1 generation but block FOXP3⁺CD4⁺CD25^(high) Tregfunction.

In the experiment of FIGS. 19A and 19B, lymphoma-derived CD4⁺CD25^(high)T cells were cultured with CFSE-labeled CD4⁺CD25^(low) cells (7×10⁴)isolated from healthy donor in the presence of irradiated allogenicmonocytes (7×10⁴, 6000 rad) and 0.3 microgram/ml anti-CD3 and 25 μg/mlof anti-hOX40 monoclonal antibody. After 3 to 4 days of culture, cellproliferation was assessed for CFSE dilution by FACS. RepresentativeFACS analyses are shown in FIG. 19A and data for all experiments areshown in FIG. 19B. We discovered that the hOX40 agonist antibodies blocklymphoma-derived CD4⁺CD25^(high) Treg function.

FIG. 20 shows the identification of OX40 agonistic antibodies that bindspecifically to human and rhesus OX40. Rhesus peripheral bloodmononuclear cells were obtained by ficoll centrifugation. CD4⁺ T cellswere obtained by CD4⁺ microbeads. CD4⁺ T cells were stimulated with 10μg/ml of lectin phaseolus vulgaris (PHA). Two days after stimulation,cells were stained with anti-hOX40 mAbs followed by goat anti-mouseIgG-APC and CD69-PE. 106-317 served as a negative control. Sixanti-hOX40 mAbs that strongly activate T cell proliferation could bindactivated rhesus CD4⁺ T cells, is shown. These results indicate that thetoxicity of these six anti-hOX40 monoclonal bodies can be tested inmonkeys.

Only seven out of 500 anti-human OX40 positive clones obtained using theconventional fusion protocols, exhibited the properties of triggeringOX40, including but not limited to, the ability to block IL-10(producing Tr1 generation and nTreg suppressive function as disclosed inTable 1.

TABLE 1 List of OX40-specific monoclonal antibodies Monoclonal antibodyclone Block IL-10 Block nTreg 1 106-108 − − 2 106-317 − − 3 106-107 − −4 106-148 − − 5 119-204A − − 6 119-220C − + 7 119-33A − + 8 119-58 − + 9119-181A − + 10 119-157A − + 11 120-140A − + 12 119-8B + − 13 119-173B +− 14 106-132 + + 15 106-222 + + 16 119-43 + + 17 119-122 + + 18119-69A + + 19 120-56 + + 20 120-270 + +

Hybridoma clones 106-222 and 119-122 were selected based on threecriteria

-   -   1. They inhibit Tr1 cell generation from CD4⁺ T cells (inducible        Treg)    -   2. They reverse the suppressive function of FOXP3⁺nTreg cells    -   3. They exhibit dose-dependent inhibition of Tr1 cells shut down        and reversal of FOXP3⁺Treg function

Chimeric and Humanized Antibodies

Humanization (also called Reshaping or CDR-grafting) is an establishedtechnique for reducing the immunogenicity of monoclonal antibodies fromxenogeneic sources (including but not limited to rodents) and forimproving their activation of the human immune system. Although themechanics of producing the engineered monoclonal antibody using thetechniques of molecular biology are known, simple grafting of the rodentcomplementary-determining regions (CDRs) into human frameworks does notalways reconstitute the binding affinity and specificity of the originalmonoclonal antibody.

In order to humanize an antibody, the design of the humanized antibodybecomes the critical step in reproducing the function of the originalmolecule. This design includes various choices: the extents of the CDRs,the human frameworks to use and the substitution of residues from therodent monoclonal antibody into the human framework regions(backmutations). The positions of these backmutations have beenidentified principally by sequence/structural analysis or by analysis ofa homology model of the variable regions' 3D structure.

Recently, phage libraries have been used to vary the amino acids atchosen positions. Similarly, many approaches have been used to choosethe most appropriate human frameworks in which to graft the rodent CDRs.Early experiments used a limited subset of well-characterized humanmonoclonal antibodies (often but not always where the structure wasavailable), irrespective of the sequence identity to the rodentmonoclonal antibody (the so-called fixed frameworks approach). Somegroups use variable regions with high amino acid sequence identity tothe rodent variable regions (homology matching or best-fit); others useconsensus or germline sequences while still others select fragments ofthe framework sequences within each light or heavy chain variable regionfrom several different human monoclonal antibodies. There are alsoapproaches to humanization developed which replace the surface rodentresidues with the most common residues found in human monoclonalantibodies (“resurfacing” or “veneering”) and those which use differingdefinitions of the extents of the CDRs. Humanized antibodies aredescribed below. However, a chimeric antibody comprising the variableheavy and light regions of SEQ ID NOs: 4 and 10, or, SEQ ID NOs: 16 and22 are also described herein.

Humanized monoclonal antibodies were be derived from the murineanti-OX40 antibody.

The isolated humanized anti-OX40 antibody may have a variable heavychain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 13. Theisolated humanized anti-OX40 antibody may have a variable heavy chainCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 14. Theisolated humanized anti-OX40 antibody may have a variable heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 15.

The isolated humanized anti-OX40 antibody may have a variable lightchain CDR1 comprising the amino acid sequence of SEQ ID NO: 7 or 19. Theisolated humanized anti-OX40 antibody may have a variable light chainCDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 20. Theisolated humanized anti-OX40 antibody may have a variable light chainCDR3 comprising the amino acid sequence of SEQ ID NO: 9 or 21.

The isolated humanized anti-OX40 antibody may have a variable lightchain comprising the amino acid sequence of SEQ ID NO: 11 or 23, or anamino acid sequence with at least 90 percent identity to the amino acidsequences of SEQ ID NO: 11 or 23. The isolated humanized anti-OX40antibody may have a variable heavy chain comprising the amino acidsequence of SEQ ID NO: 5 or 17, or an amino acid sequence with at least90 percent identity to the amino acid sequences of SEQ ID NO: 5 or 17.

The isolated humanized anti-OX40 antibody may have variable light chainencoded by the nucleic acid sequence of SEQ ID NO: 12 or 24, or anucleic acid sequence with at least 90 percent identity to the aminoacid sequences of SEQ ID NO: 12 or 24. The isolated humanized anti-OX40antibody may have variable heavy chain encoded by a nucleic acidsequence of SEQ ID NO: 6 or 18, or a nucleic acid sequence with at least90 percent identity to the amino acid sequences of SEQ ID NO: 6 or 18.

Expression of Humanized Anti-OX40 Antibodies

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel, F. M. et al. (eds.) Current Protocols in MolecularBiology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss et al

Antibodies and antibody fragments and variants can be produced from avariety of animal cells, preferably from mammalian cells, with murineand human cells being particularly preferred. Also, recombinant DNAexpression systems could include those that utilize host cells andexpression constructs that have been engineered to produce high levelsof a particular protein. Such host cells and expression constructs mayinclude Escherichia coli; harboring expression constructs derived fromplasmids or viruses (bacteriophage); yeast such as Saccharomnycescerevisieae or Pichia pastoras harboring episomal or chromosomallyintegrated expression constructs; insect cells and viruses such as Sf9cells and baculovirus; and mammalian cells harboring episomal orchromosomally integrated (including but not limited to, retroviral)expression constructs (such methods, for example, can be seen from themanuscript Verma et al., J. Immunol. Methods 216:165-181, 1998).Antibodies can also be produced in plants (such methods, for example,can be seen from U.S. Pat. No. 6,046,037; Ma et al., Science268:716-719, 1995) or by phage display technology (such methods, forexample, can be seen from Winter et al., Annu. Rev. Immunol. 12:433-455,1994).

Human anti-OX40 antibodies that displayed a level of activity andbinding specificity/affinity that are desirable can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, a VL-or VH-encoding DNA fragment is operatively linked to another DNAfragment encoding another protein, such as an antibody constant regionor a flexible linker. The term “operatively linked”, as used in thiscontext, is intended to mean that the two DNA fragments are joined suchthat the amino acid sequences encoded by the two DNA fragments remainin-frame.

In another aspect, the isolated DNA encoding the VH region can beconverted to a full-length heavy chain gene by operatively linking theVH-encoding DNA to another DNA molecule encoding heavy chain constantregions (CH1, CH2 and CH3). The sequences of human heavy chain constantregion genes are known in the art (see e.g., Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification. The heavy chain constant region can be an IgG-1,IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region and any allotypicvariant therein as described in Kabat (, Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242),but most preferably is an IgG1 or IgG4 constant region. For a Fabfragment heavy chain gene, the VH-encoding DNA can be operatively linkedto another DNA molecule encoding only the heavy chain CH1 constantregion.

Also, a humanized antibody bound to surface antigen can interact withFcR-bearing cells. Such interaction can elicit effector function such asADCC and/or enhance signaling because of Fc-mediated cross-linking. Theinteraction can be beneficial or harmful for therapy. Such harmful sideeffects include chills, fever, hypotension, and in some cases, dyspnea(Thistlethwaite J R Jr., Cosimi A B, Delmonico F L, et al.).

Certain harmful effects can originate in the protein complex found onthe surface of a T cell. Upon activation of the T cell, the proteincomplex becomes involved in the transduction of signals generated via anantigen receptor. In short, activation of the T cell starts a cascade ofevents which include the enhanced cross-linking of the antigen receptor.The cross-linking of the receptor can contribute to strong mitogenicsignaling that leads to the inducement of certain cytokines such astumour necrosis factor alpha (TNF-α), interleukin-2 (IL-2) andinterferon gamma (IFN-γ). These cytokines are known to be toxic ifgenerated in large amounts.

For example, anti-CD3 mAbs are currently used in the treatment ofautoimmune disease including as Type I diabetes mellitus in which Tcells mediated attack against pancreatic islets, producers of insulin(Kaufman A, and Herold K. Anti-CD3 mAbs for treatment of type I diabetesDiabetes Metab Res Rev 2009; 25: 302-306). Anti-CD3 antibodies are knownto inhibit lysis of targets by T cells and enhance cross-linking of theantigen receptor CD3. In addition, together with its potent mitogenicactivity, the anti-CD3 antibody is known to be a potent inducer ofcytokines, specifically, tumour necrosis factor alpha (TNF-α),interleukin-2 (IL-2) and interferon gamma (IFN-γ). The enormous releaseof cytokines, particularly TNF-α from T cells in response to the drug(Chatenoud L.) produce toxic effects. These undesirable side effectshave been attributed to the cross-linking of T cells bearing CD3molecules and the FcR bearing cells that bind to the Fc portion of theantibodies. The cross-linking activates both the T cell and the FcRbearing cells leading to the massive release of cytokines as previouslymentioned.

Similarly, potential undesirable side effects could result usinganti-OX40 antibodies. For instance, the anti-OX40 antibodies which bindto OX40 expressing T cells may also bind to FcR bearing cells andtrigger the production of cytokines that may be beneficial or harmfulfor the patients treated with the antibody. To overcome this potentialproblem, we have designed and present herein methods of mutating the FcRportion of the anti-OX40 antibodies to avoid toxics effects and providemutations to the FcR portion which may be desirable.

The site of human IgG1 that interacts with FcR (CD16, CD32 and CD64) isknown. It maps to the upper CH2 domain. The most important amino acidsare the two Leu residues at positions 234 and 235. By mutating these tworesidues to two Ala residues, interactions of IgG to all FcRs areabolished. Humanized anti-CD3 incorporated these mutations (HuOKT3AA),is a much safer drug and has a mechanism of action that is differentthan that of HuOKT3. See e.g., U.S. Pat. No. 6,491,916 incorporated byreference in its entirety herein.

The positions of the AA mutant are shown as followed:

234 235

-A-P-E-L-L-G-P-P- Wild type IgG1 upper CH2

-A-P-E-A-A-G-G-P- AA Mutant IgG1 upper CH2

Hu222AA and Hu122AA described herein may contain these mutations. If theassay system contains FcR-bearing cells, you may see the differencebetween the wild type and the AA mutant. Otherwise, the two antibodiesshould behave the same.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly.sub.4-Ser).sub.3, such that the VHand VL sequences can be expressed as a contiguous single-chain protein,with the VL and VH regions joined by the flexible linker (see e.g., Birdet al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl.Acad. Sci, USA 85:5879-5883: McCafferty et al., Nature (1990)348:552-554.

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid alterations may be introduced in the subject antibodyamino acid sequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody. Another type of amino acid variant of theantibody alters the original glycosylation pattern of the antibody. Suchaltering includes deleting one or more carbohydrate moieties found inthe antibody, and/or adding one or more glycosylation sites that are notpresent in the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 of U.S. Pat. No. 7,812,133, Col. 43, ls. 55 to Col. 44 l. 49,incorporated herein by reference, and under the heading of “preferredsubstitutions”. If such substitutions result in a change in biologicalactivity, then more substantial changes, denominated “exemplarysubstitutions” in the Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

Furthermore, substantial modifications in the biological properties ofthe antibody are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu,ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: asp,glu; (4) basic: his, lys, arg; (5) residues that influence chainorientation: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conversativesubstitutions will entail exchanging a member of one of these classesfor another class.

To express the antibodies, or antibody portions described herein, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).

As shown in FIG. 23, one such schematic structure of the expressionvector for Hu106-222 IgG1/kappa antibody. Proceeding clockwise from theSalI site at the top, the plasmid contains the heavy chain transcriptionunit starting with the human cytomegalovirus (CMV) major immediate earlypromoter and enhancer (CMV promoter) to initiate transcription of theantibody heavy chain gene. The CMV promoter is followed by the VH exon,a genomic sequence containing the human gamma-1 heavy chain constantregion including the CH1, hinge, CH2 and CH3 exons with the interveningintrons, and the polyadenylation site following the CH3 exon. After theheavy chain gene sequence, the light chain transcription unit beginswith the CMV promoter, followed by the VL exon and a genomic sequencecontaining the human kappa chain constant region exon (CL) with pa-t ofthe intron preceding it, and the polyadenylation site following the CLexon. The light chain gene is then followed by the SV40 early promoter(SV40 promoter), the E. coli xanthine guanine phosphoribosyl transferasegene (gpt), and a segment containing the SV40 polyadenylation site (SV40poly(A) site). Finally, the plasmid contains a part of the plasmidpUC19, comprising the bacterial origin of replication (pUC ori) andbeta-lactamase gene (beta lactamase). Locations of relevant restrictionenzyme sites are shown in the figure.

The recombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

As noted above, in addition to the antibody chain genes, the recombinantexpression vectors of the invention carry regulatory sequences thatcontrol the expression of the antibody chain genes in a host cell. Theterm “regulatory sequence” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals)that control the transcription or translation of the antibody chaingenes. Such regulatory sequences are described, for example, in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). It will be appreciated that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma. For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.Pat. No. 4,968,615 by Schaffner et al., U.S. Pat. No. 5,464,758 byBujard et al. and U.S. Pat. No. 5,654,168 by Bujard et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.-host cellswith methotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Mammalian hostcells for expressing the recombinant antibodies described herein includeChinese Hamster Ovary (CHO cells) (such as dhfr-CHO cells, described inUrlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, usedwith a DHFR selectable marker, e.g., as described in R. J. Kaufman andP. A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cellsand SP2 cells. When recombinant expression vectors encoding antibodygenes are introduced into mammalian host cells, the antibodies areproduced by culturing the host cells for a period of time sufficient toallow for expression of the antibody in the host cells or, secretion ofthe antibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to OX40 Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than OX40 by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods

Pharmaceutical Compositions and Pharmaceutical Administration

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). The compositions of this invention may be in a varietyof forms. These include, for example, liquid, semi-solid and soliddosage forms, such as liquid solutions (e.g., injectable and infusiblesolutions), dispersions or suspensions, tablets, pills, powders,liposomes and suppositories. The preferred form depends on the intendedmode of administration and therapeutic application. Typical compositionsare in the form of injectable or infusible solutions, such ascompositions similar to those used for passive immunization of humanswith other antibodies. The antibody can be administered by intravenousinfusion or injection or intramuscular or subcutaneous injection.

The route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is co-formulated with and/or co-administered with one ormore additional therapeutic agents that are useful for treatingdisorders in which OX40 inactivation is detrimental. For example, ananti-OX40 antibody or antibody portion of the invention may beco-formulated and/or co-administered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind othercytokines or that bind cell surface molecules). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies. It will be appreciated by the skilledpractitioner that when the antibodies of the invention are used as partof a combination therapy, a lower dosage of antibody may be desirablethan when the antibody alone is administered to a subject (e.g., asynergistic therapeutic effect may be achieved through the use ofcombination therapy which, in turn, permits use of a lower dose of theantibody to achieve the desired therapeutic effect.

Antibodies described herein, or antigen binding portions thereof can beused alone or in combination to treat such diseases. It should beunderstood that these antibodies or antigen binding portion thereof canbe used alone or in combination with an additional agent, e.g., atherapeutic agent, said additional agent being selected by the skilledartisan for its intended purpose. For example, the additional agent canbe a therapeutic agent art-recognized as being useful to treat thedisease or condition being treated by the antibody taught herein. Theadditional agent also can be an agent which imparts a beneficialattribute to the therapeutic composition e.g., an agent which effectsthe viscosity of the composition.

The pharmaceutical compositions described herein may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may vary according to factors such as the diseasestate, age, sex, and weight of the individual; and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic or prophylacticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

Example I

Chimeric and humanized 106-222 IgG1/kappa monoclonal antibodies (Ch222and Hu222, respectively) were purified from culture supernatants of thecorresponding NS0 stable transfectants using a protein A column asdescribed in Appendices A and B. Hu222 was eluted from the column by twodifferent ways. Briefly, Hu222 Lot I was eluted with low pH buffer andLot II with Pierce's Gentle Ag/Ab Elution Buffer. The yield of Hu222 wasbetter when the low pH buffer was used for elution. Ch222 was elutedfrom the column with Gentle Ag/Ab Elution Buffer.

Purified Hu222 Lot I and II antibodies were characterized by SDS-PAGEalongside with mouse 106-222 according to standard procedures. Five μgof each antibody was analyzed under reducing conditions. As shown inFIG. 21, each of Hu222 Lot I and II antibodies is comprised of a heavychain with a molecular weight of about 50 kD and a light chain with amolecular weight of about 25 kD. The purity of Hu222 Lot I and IIantibodies appeared to be more than 95%.

Endotoxin contamination in the humanized antibodies was analyzed withLonza's Limulus Amebocyte Lysate (LAL) QCL-1000 kit. The endotoxin levelwas less than 0.5 EU/mg protein for both Hu222 Lot I and II antibodies.

Characterization of Hu106-222 for Binding to L/OX40 Cells

Binding of mouse 106-222, Ch106-222 and Hu106-222 antibodies to OX40 wasexamined in a FACS binding assay with L/hOX40 cells essentiallyaccording to the protocol supplied by Dr. Laura Bover. Antibodies boundto L/hOX40 cells were detected with PE-labeled goat anti-mouse IgGantibody (for mouse 106-222) or PE-labeled goat anti-human IgG antibody(for Ch106 and Hu106).

FIG. 22 shows the analysis of mouse 106-222, Ch106 and Hu106-222 (LotII) antibodies for binding to L/OX40 cells. The titration curve ofHu106-222 (Lot II) was nearly identical to that of Ch106-222, indicatingthat the antigen binding affinity of mouse 106-222 is retained inHu106-222. The titration curve of mouse 106-222 was similar to those ofCh106 and Hu106; however, due to the difference of the secondaryantibodies, the data only indicates that the affinity of mouse 106-222is similar to that of Hu106-222.

FIG. 24 shows the comparison between Hu106-222 Lot I and II antibodiesfor binding to L/hOX40 cells. Although further analysis is needed, theaffinity of the two lots of Hu106-222 appeared to be similar, if notidentical, to each other. Hence, acid elution of Hu106-222 from aprotein A column does not seem to affect its affinity.

Purification of Ch106-222

NS0 stable transfectant C8 was grown in 500 ml of Invitrogen's HybridomaSFM medium in a roller bottle to exhaustion. The culture was spun downin Corning's 250 ml Centrifuge Tube (Cat #430776) in Beckman Coulter'sAllegra X-12R Centrifuge (2000 RPM for 15 min). The culture supernatantwas loaded onto a 1 ml GE Healthcare HiTrap MabSelect SuRe column (Cat#11-034-95) using a Pharmacia P1 pump. The column was washed withTris-buffered saline (Pierce, Cat #28379) and eluted with Pierce'sGentle Ag/Ab Elution Buffer (Cat #21027). Fractions (about 1 ml) werecollected and their OD at 280 nm read.

Fraction # OD at 280 nm 3 0.12 4 0.30 5 0.18 6 0.11

Fractions 3 to 6 were pooled (volume=3.0 ml, OD at 280 nm=0.14). Pooledfractions were desalted onto a 10 ml Sephadex G25 medium column in PBS.Fractions of 1 ml were collected.

Fraction # OD at 280 nm 5 0.09 6 0.19 7 0.12 8 0.12 9 0.00

Fractions 6 to 9 were pooled (volume=3.0 ml, OD at 280 nm=0.11). Pooledfractions were dialyzed overnight in PBS. After dialysis, the volume was3.0 ml and OD at 280 nm was 0.19. This preparation is called Ch106, lotAug. 31, 2009, with a concentration of 0.13 mg/ml.

Purification of Hu106-222

NS0 stable transfectant 1-C6 was grown in 500 ml of Invitrogen'sHybridoma SFM medium in a roller bottle to exhaustion. The culture wasspun down in Corning's 250 ml Centrifuge Tube (Cat #430776) in BeckmanCoulter's Allegra X-12R Centrifuge (2000 RPM for 15 min).

Lot 1: 150 ml of the culture supernatant was loaded onto a 1 ml GEHealthcare HiTrap MabSelect SuRe column (Cat #11-034-95) using aPharmacia P1 pump. The column was washed with PBS and bound antibody waseluted with 0.1M glycine-HCl, 0.1 M NaCl (pH 3.0). Eluted fractions (1ml each) were collected into tubes containing 50 μl 1M Tris-HCl (pH8.0).

Fraction # OD at 280 nm 2 0.88 3 2.84 4 1.29 5 0.63 6 0.18

Fractions 2 to 5 were pooled (volume=4.2 ml, OD at 280 nm=1.59). Pooledfractions were dialyzed overnight in PBS. After dialysis, the volume was4.2 ml and OD at 280 nm was 1.54. The antibody solution (lot Sep. 18,2009 I; 1.1 mg/ml) was filter-sterilized.

Lot II:

The remaining culture supernatant (350 ml) was loaded onto a 1 ml GEHealthcare HiTrap MabSelect SuRe column using a Pharmacia P1 pump. Thecolumn was washed with Tris-buffered saline and eluted with Gentle Ag/AbElution Buffer. Fractions (about 1 ml) were collected and their OD readat 280 nm.

Fraction # OD at 280 nm 2 0.12 3 0.85 4 2.17 5 1.47 6 1.02 7 0.81 8 0.669 0.54 10 0.44 11 0.46

Fractions 3 to 7 were pooled (volume=4.2 ml, OD at 280 nm=1.22). Thecolumn was washed again with Tris-buffered saline and antibody elutedwith 0.1M glycine-HCl, 0.1M NaCl (pH 3.0) to examine if elution byGentleAg/Ab Elution Buffer was efficient.

Fraction # OD at 280 nm 1 0.05 2 0.05 3 1.23 4 0.49 5 0.10

Fractions 3 to 7 eluted with GentleAg/Ab Elution Buffer were pooled anddesalted onto a 10 ml Sephadex G25 medium column in PBS. Fractions of 1ml were collected.

Fraction # OD at 280 nm 4 0.38 5 0.96 6 1.38 7 1.33 8 1.10 9 0.12

Fractions 5 to 8 were pooled (volume=4.0 ml, OD at 280 nm=1.12). Pooledfractions were dialyzed overnight in PBS. After dialysis, the volume was4.0 ml and OD at 280 nm was 1.12. The antibody solution (lot Sep. 18,2009 II; 0.8 mg/ml) was filter-sterilized.

The high salt elution method with Pierce's Gentle Ag/Ab Elution Bufferwas not as efficient as the low pH method to elute bound human IgG1antibody from the protein A column. As antibodies were not eluted in asharp peak with Gentle Ag/Ab Elution Buffer, it was necessary to poolmany fractions for collection of eluted IgG and desalt the pooledfractions before dialysis. The poor elation profile with Gentle Ag/AbElution Buffer and the extra purification step affected the yield ofantibody. It is advised that the high salt elution method is used onlyif IgG to be purified is acid labile.

Example II Purification of Ch119-122 and Hu119-122 Antibodies

Chimeric 119-122 IgG1/kappa monoclonal antibody (Ch119) was purifiedfrom culture supernatant of the corresponding NS0 stable transfectant(clone G11) grown in Hybridoma-SFM media (Invitrogen) using a protein Acolumn. After elution with Pierce's Gentle Ag/Ab Elution Buffer, thebuffer of Ch119 was exchanged to PBS by gel filtration and thendialysis. The concentration of Ch119 was 0.21 mg/ml.

Humanized 119-122 IgG1/kappa monoclonal antibody (Hu122) was purifiedfrom culture supernatant of the corresponding NS0 stable transfectant(clone 2F5) grown in Hybridoma-SFM media using a protein A column.Hu106-222 was eluted from the column with low pH buffer, neutralizedwith 1 M Tris-HCl (pH 8.0), and dialyzed in PBS. The concentration ofHu122 was 1.6 mg/ml.

Purified Hu106-222 was characterized by SDS-PAGE alongside with mouse119-122 according to standard procedures. Five μg of each antibody wasanalyzed under reducing conditions. As shown in FIG. 25, Hu119-122 iscomprised of a heavy chain with a molecular weight of about 50 kD and alight chain with a molecular weight of about 25 kD. The purity of Hu119appeared to be more than 95%.

Characterization of Hu119-122 for Binding to L/hOX40 Cells

Binding of mouse 119-122, Ch119-122 and Hu119-122 antibodies to OX40 wasexamined in a FACS binding assay with L/OX40 cells essentially accordingto the protocol supplied by Dr. Laura Bover. Antibodies bound to L/OX40cells were detected with PE-labeled goat anti-mouse IgG antibody (formouse 119-122) or PE-labeled goat anti-human IgG antibody (for Ch119-122and Hu119-122).

FIG. 26 shows the result of the FACS analysis. The titration curve ofHu119-122 was similar to that of Ch119-122, suggesting that the antigenbinding affinity of mouse 119-122 is retained in Hu119-122. However, theMCF values at higher antibody concentrations of Ch109-122 and Hu119-122do not fall right on the corresponding curves. After adjusting theexperimental conditions, the FACS analysis should be repeated.

Example III

To evaluate the ability of our humanized anti-human OX40 antibodies toenhance T cell proliferation, we performed proliferation assays usinganti-CD3 coated CD32-L cells and freshly sorted naïve CD4⁺ T cells. FIG.27 shows that humanized anti-human OX40 mAb clone 119-122 (Hu122), andits FcR binding mutated antibody (Hu122-AA) enhanced naïve CD4⁺ T cellproliferation. Hu122 yielded better T cell stimulatory activity comparedto parental mouse anti-human OX40 mAb (Mouse122). (FIG. 27)

FcR binding mutated humanized anti-human OX40 mAb clone 106-222(Hu222-AA) and chimeric anti-human OX40 mAb clone 106-222 (Ch222)enhanced anti-CD3 stimulated naïve CD4⁺ T cell proliferation. Theseantibodies have similar stimulatory activity compared to parental mouseanti-human OX40 mAb (Mouse106-222). However, the fully humanizedanti-human OX40 Ab, Hu106, did not enhance T cell proliferation. (FIG.28)

To evaluate the ability of humanized anti-human OX40 antibodies to blockCD4⁺ regulatory T cell (Tregs) suppressive function, we performedproliferation assays using freshly sorted naïve CD4⁺ T cells andCD4⁺CD25^(high)CD127^(low) Tregs. We found that the chimeric antibodyCh122 and Fc binding mutated humanized antibody (Hu122-AA) exhibitedbetter potency than parental mouse anti-human OX40 mAb (Mouse122) inblocking CD4⁺ Treg suppressive function. (FIG. 29 A-B)

In the experiment of FIG. 27, freshly sortedCD4⁺CD25^(low)CD127⁺CD45RO⁻CD45RA⁺ naïve T cells were stimulated with Lcells expressing CD32 (CD32-L) coated with 4 concentrations of anti-CD3antibodies plus 2 μg/ml of anti-human OX40 Ab clone 119 antibodies orcontrol antibodies. Three days after stimulation, radioisotope tritiumwas added and cultured for additional 16-18 hrs before cell harvest.Data are a representative of experiments from two donors. CD32-Lcells-expressing hOX40 ligand (CD32-L/hOX40L) serves as positivecontrol, while human and mouse IgG1 serve as negative controls.

In the experiment of FIG. 28, freshly sorted naïve CD4⁺ T cells werestimulated with CD32-L cells coated with 4 concentrations of anti-CD3antibodies plus 2 μg/ml of anti-human OX40 mAb clone 106-222 (Hu222)antibodies or control antibodies. Three days after stimulation,radioisotope tritium was added and cultured for additional 16-18 hrsbefore cell harvest. Data are representative of experiments from twodonors. CD32-L/hOX40L serves as positive control, while human and mouseIgG1 serve as negative controls.

In the experiment of FIG. 29, freshly sorted CD4⁺ naïve T cells werecultured in the presence of CD4⁺CD25^(high)CD127^(low)Tregs at threeTregs: T effector ratios and were stimulated with CD32-L cells coatedwith 0.2 μg/ml of anti-CD3 antibodies plus 10 μg/ml of anti-human OX40mAb clone 119-122 antibodies or control antibodies. Three days afterstimulation, radioisotope tritium was added and cultured for additional16-18 hrs before cell harvest. Data are representative of threeexperiments. CD32-L/hOX40L serves as positive control, while human andmouse IgG1 serve as negative controls.

Example IV

Since antibodies will encounter total peripheral blood mononuclear cells(PBMCs) when they are given to patients via intravenous injection, wetested the ability of our anti-human OX40 antibodies to stimulate T cellproliferation using PBMCs as antigen-presenting cells (APCs) in ourproliferation assays. However, we obtained highly variable data with ourmouse anti-human OX40 mAbs when using PBMCs as APCs that is not seenwhen using monocytes as APCs, suggesting that our antibodies requiresome kind of cross-linking for activity. To test this possibility,plates were coated with our anti-human OX40 mAbs and anti-CD3, washed,and used to stimulate CD4⁺ or CD8⁺ T cell proliferation in the absenceof accessory cells. FIG. 30 shows the results that anti-human OX40antibodies enhance CD4⁺ and CD8⁺ T cell proliferation.

Freshly sorted 1×10⁵ of CD4⁺CD25^(low)CD45RO⁻CD45RA⁺ naïve T cells (FIG.30A) or CD3⁺CD8⁺ T cells (FIG. 30B) were stimulated with plate-boundanti-CD3 (3 μg/ml) and anti-human OX40 mouse mAb (2 μg/ml). Tritiatedthymidine was added on the third day of culture and cells were harvestedafter another 15 hours of incubation. Proliferation of T cells wasevaluated by thymidine incorporation. Anti-human OX40 mAbs were derivedfrom three hybridoma fusions. Numbers following fusion number denote aspecific antibody. Mouse IgG1 and 119-42 served as negative controls.Each treatment was performed in triplicate. Representative data from 4 Tcell donors are shown. (FIG. 30C) All three versions of humanizedanti-human OX40 mAbs [Hu106-222 and Hu119-122; Hu106-222AA andHu119-122AA (AA denotes two of the Fc binding residues were mutated tothe amino acid alanine); and Ch119-122 (similar to humanized 119-122except that the mouse variable region “paratope” was maintained)]stimulated naïve CD4⁺ T cell proliferation. Anti-CD28 served as apositive control.

As shown in FIG. 30, panels A and B, show that plate-bound mouseanti-human OX40 mAbs potently stimulated proliferation of naïve CD4⁺ Tcells and CD8⁺ T cells by a range of 10 to 40 fold. We extended ourstudies to our humanized anti-human OX40 mAbs and found that the threeversions of our humanized antibodies, whether it was fully humanized,chimeric or had AA mutants in which residues responsible for binding tothe Fc receptor were altered to alanine, were potent stimulators ofnaïve CD4⁺ T cell proliferation (FIG. 30C).

FIG. 31 shows mouse and humanized anti-human OX40 antibodies requirecross-linking in order to enhance T cell proliferation. Freshly sortednaïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 (3 μg/ml)plus plate-bound or soluble humanized anti-human OX40 mAbs (2 μg/ml) inthe absence of accessory cells. Tritiated thymidine was added on thethird day of culture and cells were harvested after another 15 hours ofincubation. Proliferation of T cells was evaluated by thymidineincorporation. Mouse IgG1 and anti-CD28 served as negative and positivecontrols, respectively. Representative data from two donors are shown.Naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 in theabsence of accessory cells. The next day, anti-human OX40 mAb 119-122 (2μg/ml) was added alone or in combination with equal amount of asecondary antibody against Fc. Cell proliferation was evaluated asdescribed in panel A.

The potency of our humanized anti-human OX40 mAbs Hu106-222 andHu119-122 was comparable to that of anti-CD28. In contrast, when solubleanti-human OX40 antibody was added to the T cell culture, thestimulatory effect was abolished. (FIG. 31A). However, when solubleanti-human OX40 mAb 119-122 was added together with a F(ab′)₂ fragmentgoat anti-mouse IgG, Fc fragment specific secondary antibody, thestimulatory effect was restored (FIG. 31B). These results demonstratethat anti-human OX40 mAbs require cross-linking for their biologicalactivities.

To evaluate the ability of our agonistic, anti-human OX40 mAbs to blockthe suppressive function of CD4⁺CD25^(low)CD12T nTregs, we performedproliferation assays in the presence of CD4⁺CD25^(low)CD127+CD45RO+ Teffector cells (Teff) and CD4⁺ nTregs. By using our plate-bound systemin which anti-human OX40 mAbs together with anti-CD3 were coated on aplate and in the absence of accessory cells, twelve (222, 132, 8B, 33A,43, 58B, 122, 157A, 173B, 220C, 140A, 270) of our anti-human OX40 mousemAbs potently inhibited nTreg suppression (FIGS. 32A and 32B). Althoughthe ratio of nTregs to T effector cell used in these assays was 1:1,these antibodies were able to stimulate T effector cells to proliferate10 to 35 percent above the percentage achieved by T effector cells inthe absence of nTregs. Our humanized anti-human OX40 mAbs also reversedthe suppressive function of nTregs at similar levels (FIG. 32C). Theseresults taken together suggest that our anti-human OX40 mouse mAbs arepotent stimulators of OX40, resulting in significant enhancement of Tcell proliferation and inhibition of nTreg suppressive function.Furthermore, our humanized anti-human OX40 mAbs maintained the potentbiological activities of their parental mouse antibodies.

FIG. 32 shows anti-human OX20 mAb block the activity ofCD4⁺FOXP3⁺nTregs. CFSE-labeled CD4⁺CD25⁻CD45RO⁺ T effector cells andCD4⁺FOXP3⁺Tregs were derived from the same healthy donor. T cells werestimulated with soluble anti-CD28 (0.5 μg/ml) and plate-bound anti-CD3(3 (3 μg/ml) and anti-human OX40 mAbs (2 μg/ml). Proliferation of Teffector cells was evaluated by flow cytometry for CFSE dilution. Theratio of nTregs to T effector cells was 1:1. Mouse IgG1 served asnegative control. Naïve CD4⁺ T cells served as control T cells todemonstrate specific inhibition of T effector cell proliferation bynTregs. FIG. 32A is a representative FACS data showing the proliferationof T effector cells in the presence of naïve CD4⁺ T cells, nTregs ornTregs plus the anti-human OX40 mAb 119-33A. FIG. 32B shows thepercentage of T effector cell proliferation in the presence of nTregsafter treatment with a mouse anti-human OX40 mAb (20 tested). FIG. 32Cshows all three versions of humanized anti-human OX40 mAbs restoredproliferation of T effector cells.

A recent report suggests that OX40 triggering can induce apoptosis of ahuman T cell line expressing OX40 (Yoshiaki Takahashi et al., 200B, AidsResearch and human Retroviruses, 24). We therefore tested the effect ofincreasing concentrations of the anti-human OX40 mAb 106-222 plus afixed, low dose of anti-CD3 on the survival of three T cell subsets inthe presence of monocytes. FIG. 33A shows that high concentrations ofanti-human OX40 mAb 106-222 (20-30 μg/ml) preferentially killedactivated FOXP3⁺ nTregs while activated naïve and memory CD4⁺ Tcellswere either resistant or less susceptible to this effort. To testwhether the anti-human OX40 mAb acts directly on Tregs to induce celldeath, we performed new experiments in the absence of accessory cells.FIG. 33B shows that strong OX40 signaling in combination with anti-CD3specifically killed nTregs in the absence of accessory cells. To confirmif the killing effects mediated by anti-human OX40 mAb mimicked OX40triggering by natural OX40 ligand, we used a mouse fibroblast L cellline that over-expressed hOX40L and used it to stimulate nTregs in thepresence of a low dose of anti-CD3 and obtained similar killing effectson nTregs (FIG. 33C). These results suggest that strong OX40 triggeringkills OX40-expressing Tregs cells.

Specifically, FIG. 33 shows high concentration of anti-human OX40 mAbpreferentially kills FOXP3⁺ Tregs. In FIG. 33A, T cell subsets (naïve,CD4⁺CD25^(low)CD127⁺CD45RO⁻CD45RA⁺; memory, CD4⁺CD25^(low)CD127⁺CD45RA⁻CD45 RO⁺; and nTregs, CD4⁺CD25^(high)CD127^(low)) were each culturedwith an equal ratio of CD14⁺ monocytes in the presence of solubleanti-CD3 (0.3 μg/ml) and increasing concentrations of the mouseanti-human OX40 mAb 106-222. Cell viability was determined after 3 daysof culture by flow cytometry analysis, gating on viable lymphocytes.Data, from two T cell donors are shown. FIGS. 33B and 33C show strongtriggering of OX40 kills CD4⁺FOXP3⁺Tregs. FIG. 33B shows that CD4⁺FOXP3⁺Tregs were stimulated with plate-bound anti-CD3 (2 μg/ml) plus soluble119-122 mAb (30 μg per million cells) or mouse IgG1 control antibody.Trypan blue-negative live cells after one day of culture were countedwith a hemacytometer. FIG. 33C shows that CD4⁺FOXP3⁺ Tregs werestimulated with soluble anti-CD3 (0.2 μg/ml) plus L cells or L cellsexpressing the hOX40 ligand (L/hOX40L). Live cells were counted afterone day of stimulation.

We next sought to determine whether anti-human OX40 mAb acts directly onT cells to block nTreg suppressive function. Freshly sorted CD4⁺ Teffector cells or nTregs were preactivated overnight with anti-CD3 andthen pulsed with anti-human OX40 mAbs for 4 hours. T effectors cellswere then washed, labeled with CFSE, and co-cultured with nTregs in thepresence of an equal number of CD14⁺ monocytes and anti-CD3. Similarly,the pre-stimulated nTregs were washed and cultured with untreatedCFSE-labeled T effector cells.

FIG. 34 shows anti-human OX40 mAbs act directly on T cells to block thesuppressive function of Tregs. FIG. 34A shows anti-human OX40 mAb actsdirectly on effector memory T cells to confer them resistant tosuppression by nTregs. CD4⁺ CD25^(low)CD127⁺CD45RA⁻ CD45RO⁺ memory Tcells were stimulated with plate-bound anti-CD3 (0.8 μg/ml) in culturemedium (RPMI/10% FCS/P/S plus 1 L-2 at 30 IU/ml) for 12 hours, thenpulsed with anti-human OX40 mAb (119-122, 22 μg per 0.5 million cells)in culture medium for 4 hours, washed 3 times, and 8×10⁴ of CFSE-labeledeffector T cells were cultured with decreasing ratios of nTregs.Proliferation of effector T cells was evaluated by flow cytometry forCFSE dilution. Anti-human OX40 mAb acts on Tregs making them unable tosuppress T effector cell proliferation (FIG. 34B).CD4⁺CD25^(high)CD127^(low) nTregs were pre-stimulated with plate-boundanti-CD3 (2 μg/ml) in culture medium for 12 hours, then pulsed with ananti-human OX40 mAb, 119-122 or 106-222, or a control antibody,anti-ICOS or mouse IgG1, as described in panel A, washed and culturedwith CFSE-labeled T effector memory cells. Proliferation of T effectorcells was evaluated by flow cytometry for CFSE dilution.

T effector cells treated with anti-human OX40 mAb became resistant tosuppression by nTreg cells. (FIG. 34A) By contrast, proliferation of Teffector cells treated with mouse IgG1 control antibody remainedsusceptible to suppression by nTregs. FIG. 34B shows that nTregs treatedwith anti-human OX40 mAbs were unable to suppress proliferation of Teffector cells. By contrast, nTregs treated with control antibodies,such as anti-I COS or mouse IgG1, remained suppressive. These resultssuggest that our anti-human OX40 mAbs act directly on both T effectorcells and nTregs to restore T effector cell proliferation.

Example V

Supplemental preliminary in vivo data showed that anti-human OX40antibody works in mice enhances T cell expansion and tumor rejection inmice. It was previously shown that anti-human OX40 mAb can specificallyactivate the NF-κB cascade in mouse CD8⁺ T cells transduced with humanOX40. To determine whether the anti-hOX40 mAb can enhance tumorrejection by promoting effector CD8⁺ T cell survival and clonalexpansion in vivo, transgenic Pmel CD8⁺ T cells transduced with theluciferase gene and hOX40 were adaptively transferred into C57BL/6albino mice bearing non-pigmented MC38 tumors. After adoptive transferof the transduced T cells, mice were treated with Abs. It was found thatsignificantly more human OX40⁺ luciferase+Pmel T cells migrated into thelung on day 4 in mice treated with anti-hOX40 mAb compared with mousetreated with IgG1 control antibody (FIG. 35B), indicating that hOX40triggering in mice promoted CD8⁺ T cell expansion. Upon day 8 (data notshown) and day 12 after treatment, it was found that the same group ofmice treated with anti-hOX4 mAb retained significantly moreluciferase⁺Pmel T cells at the tumor site compared with the controlgroup of mice treated with IgG1 (FIG. 35B), again indicating that hOX40triggering in mice promoted CD8⁺ T cell survival. Finally, tumor sizesof mice that received hOX40⁺Pmel CD8⁺ T cells and subsequently treatedwith anti-hOX40 mAb were significantly smaller compared with those ofmice that received nontransduced Pmel T cells and treated withanti-hOX40 mAb or hOX40-Pmel T cells followed by treatment with controlmouse IgG1-match antibody. These results show that the triggering ofhuman OX40 in mice results in biological effects similar to those ofmouse OX40 (Gough M J et, 2008). Therefore, the data demonstrates theability of anti-human OX40 mAb to stimulate CD⁺Tcell expansion andsurvival in vivo and enhance tumor rejection.

Anti-Human OX40 mAb Promotes T Cell Expansion and Survival In Vivo.

Our therapeutic vaccination regimen is shown in FIG. 35A. C57BL/6 albinomice in groups of 5 were subcutaneously (S.C) implanted with 5×10⁵non-pigmented MC38/gp100 tumor cells (day 0). On day 6, lymphopenia wasinduced by administering a 350 cGy dose of radiation. On day 7, 1×10⁶luciferase transduced Pmel-1 T cells with or without human OX40expression were adoptively transferred into tumor-bearing mice (n=5 pergroup), followed by intravenous injection of 5×10⁵ Gp100 peptide-pulsedDCs. Recombinant human IL-2 was intraperitoneally administered for 3 dafter T cell transfer. Antibodies were administered on days 7, 9 and 11with 100, 50 and 50 μg, respectively, per injection per mouse (FIG.35B). In vivo bioluminescence images showed accumulation ofluciferase-expressing CD8⁺ pmel-1 T cells in the lung and tumor sites ondays 4 and 12. Two of five mice per group on day 4 and day 12 are shown(FIG. 35C). Tumors responded to treatments using anti-hOX40 mAb. Tumorsize was measured every 3 days. Pmel-1 and Pmel-1 plus mouse IgG1 servedas controls.

We claim:
 1. An isolated antibody or antigen-binding portion thereofwhich binds to OX40 comprising: (a) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:1; (b) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:2;(c) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO:3; (d) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:7; (e) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:8;and (f) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO:9.
 2. An isolated antibody or antigen-bindingportion thereof according to claim 1, wherein the isolated antibodycomprises a light chain variable region having a sequence at least 90%identical to the amino acid sequence of SEQ ID NO:10 and a heavy chainvariable region having a sequence at least 90% identical to the aminoacid sequence of SEQ ID NO:4.
 3. The isolated antibody orantigen-binding portion thereof of claim 2, wherein the isolatedantibody comprises a light chain variable region having the amino acidsequence of SEQ ID NO:10 and a heavy chain variable region having theamino acid sequence of SEQ ID NO:4.
 4. The isolated antibody orantigen-binding portion thereof of claim 1, comprising a light chainvariable region having a sequence at least 90% identical to the aminoacid sequence of SEQ ID NO:11 and a heavy chain variable region having asequence at least 90% identical to the amino acid sequence of SEQ IDNO:5.
 5. The isolated of antibody or antigen-binding portion thereof ofclaim 4, comprising a light chain variable region having the amino acidsequence of SEQ ID NO:11 and a heavy chain variable region having theamino acid sequence of SEQ ID NO:5.
 6. An isolated antibody orantigen-binding portion thereof of claim 1, wherein the isolatedantibody is a monoclonal antibody.
 7. The isolated antibody orantigen-binding portion thereof of claim 6, wherein the isolatedmonoclonal antibody is a humanized antibody.
 8. An antigen-bindingportion of an antibody according to claim 1, wherein the antigen-bindingportion retains the ability to specifically bind and agonize OX40receptor.
 9. A pharmaceutical composition comprising the antibody orantigen-binding portion thereof of claim 1.